Antimicrobial agents

ABSTRACT

The present invention relates to endolysin variants comprising an endolysin to which a peptide stretch with membrane or LPS disrupting activity is fused. Moreover, the present invention relates to nucleic acid molecules encoding said modified endolysin variant, vectors comprising said nucleic acid molecules and host cells comprising either said nucleic acid molecules or said vectors. In addition, the present invention relates to a method for producing said endolysin variant. Further, the present invention relates to said modified endolysin variant for use as a medicament, in particular for the treatment or prevention of Gram-negative bacterial infections, as diagnostic means, disinfectant or as cosmetic substance. The present invention also relates to the removal or reduction or prevention of Gram-negative bacterial contamination of foodstuff, of food processing equipment, of food processing plants, of surfaces coming into contact with foodstuff, of medical devices, of surfaces in hospitals and surgeries. Furthermore, the present invention relates to the use of said endolysin variant as a diagnostic means in medicinal, food or feed or environmental diagnostic. Finally, the present invention relates to a pharmaceutical composition comprising said modified endolysin variant.

This application is a national phase application under 35 U.S.C. §371 ofInternational Patent Application No. PCT/EP2009/060947 filed Aug. 25,2009 which claims priority to Great Britain Patent Application No. GB0815484.1 filed Aug. 26, 2008. The entire text of each of theabove-referenced disclosures are specifically incorporated herein byreference without disclaimer.

The present invention relates to modified endolysin variants withimproved antibacterial action against Gram-negative bacteria. Saidmodified endolysin variants comprise an endolysin and a cationic peptidefused to the endolysin, thus enhancing the cationicity of saidendolysin. The present invention also relates to a microorganismtransformed with a nucleic acid comprising a nucleotide sequenceencoding a modified endolysin variant with enhanced cationicity. Theinvention further relates to a method for producing an endolysin variantusing a microorganism transformed with a nucleic acid encoding anendolysin variant according to the present invention as productionorganism.

In particular the present invention relates to endolysin variantscomprising an endolysin to which a peptide stretch with membrane or LPSdisrupting activity is fused. Moreover, the present invention relates tonucleic acid molecules encoding said modified endolysin variant, vectorscomprising said nucleic acid molecules and host cells comprising eithersaid nucleic acid molecules or said vectors. In addition, the presentinvention relates to a method for producing said endolysin variant.Further, the present invention relates to said modified endolysinvariant for use as a medicament, in particular for the treatment orprevention of Gram-negative bacterial infections, as diagnostic means,disinfectant or as cosmetic substance. The present invention alsorelates to the removal or reduction or prevention of Gram-negativebacterial contamination of foodstuff, of food processing equipment, offood processing plants, of surfaces coming into contact with foodstuff,of medical devices, of surfaces in hospitals and surgeries. Furthermore,the present invention relates to the use of said endolysin variant as adiagnostic means in medicinal, food or feed or environmental diagnostic.Finally, the present invention relates to a pharmaceutical compositioncomprising said modified endolysin variant.

Endolysins are peptidoglycan hydrolases encoded by bacteriophages (orbacterial viruses). They are synthesized during late gene expression inthe lytic cycle of phage multiplication and mediate the release ofprogeny virions from infected cells through degradation of the bacterialpeptidoglycan. They are either β(1,4)-glycosylases (lysozymes),transglycosylases, amidases or endopeptidases. Antimicrobial applicationof endolysins was already suggested in 1991 by Gasson (GB2243611).Although the killing capacity of endolysins has been known for a longtime, the use of these enzymes as antibacterials was ignored due to thesuccess and dominance of antibiotics. Only after the appearance ofmultiple antibiotic resistant bacteria this simple concept of combatinghuman pathogens with endolysins received interest. A compelling need todevelop totally new classes of antibacterial agents emerged andendolysins used as ‘enzybiotics’—a hybrid term of ‘enzymes’ and‘antibiotics’—perfectly met this need. In 2001, Fischetti and coworkersdemonstrated for the first time the therapeutic potential ofbacteriophage Cl endolysin towards group A streptococci (Nelson et al.,2001). Since then many publications have established endolysins as anattractive and complementary alternative to control bacterialinfections, particularly by Gram-positive bacteria. Subsequentlydifferent endolysins against other Gram-positive pathogens such asStreptococcus pneumoniae (Loeffler et al., 2001), Bacillus anthracis(Schuch et al., 2002), S. agalactiae (Cheng et al., 2005) andStaphylococcus aureus (Rashel et al, 2007) have proven their efficacy asenzybiotics. Nowadays, the most important challenge of endolysin therapylies in the insensitivity of Gram-negative bacteria towards theexogenous action of endolysins, since the outer membrane shields theaccess of endolysins from the peptidoglycan. This currently prevents theexpansion of the range of effective endolysins to importantGram-negative pathogens.

Gram-negative bacteria possess an outer membrane, with itscharacteristic asymmetric bilayer as a hallmark. The outer membranebilayer consists of an inner monolayer containing phospholipids(primarily phosphatidyl ethanolamine) and an outer monolayer that ismainly composed of a single glycolipid, lipopolysaccharide (LPS). Thereis an immense diversity of LPS structures in the bacterial kingdom andthe LPS structure may be modified in response to prevailingenvironmental conditions. The stability of the LPS layer and interactionbetween different LPS molecules is mainly achieved by the electrostaticinteraction of divalent ions (Mg²⁺, Ca²⁺) with the anionic components ofthe LPS molecule (phosphate groups in the lipid A and the inner core andcarboxyl groups of KDO). Therefore, the cation-binding sites areessential for the integrity of the outer membrane (Vaara, 1992).Polycationic agents such as poly-L-lysine polymers (of at least 20residues) increase the outer membrane permeability by displacement ofthese stabilizing divalent cations. In addition, they exert a so-called‘self-promoted uptake’ mechanism (Hancock and Wong, 1984). Due to theirbulkiness, they disrupt the normal barrier function of the outermembrane and create transient cracks, promoting their own uptake (Vaaraand Vaara, 1983). Furthermore, the dense and ordered packing of thehydrophobic moiety of lipid A, favored by the absence of unsaturatedfatty acids, forms a rigid structure with high viscosity. This makes itless permeable for lipophilic molecules and confers additional stabilityto the outer membrane (OM).

Increasingly microbial resistance to antibiotics, however, is creatingdifficulties in treating more and more infections caused by bacteria.Particular difficulties arise with infections caused by Gram-negativebacteria like Pseudomonas aeruginosa and Enterobacteriaceae.

Thus, there is a need for new antimicrobial agents against Gram-negativebacteria.

This object is solved by the subject matter defined in the claims.

The following figures illustrate the present invention.

FIG. 1 is a schematic overview showing plasmid construction forrecombinant production of (POLY)^(n)-gp144 ((POLY)^(n)-KZ144).Previously, pEXP5CT/POLY-gp144 (pEXP5CT/POLY-KZ144) was constructed by atail PCR (with the BamHI restriction site and first polycation cassettein the 5′ tail primer). The plasmid was linearized with BamHI,dephosphorylated and ligated with a cassette containing overhangingBamHI ends. This cassette originates from the hybridization of twocomplementary oligonucleotides and encodes 9 positively chargedresidues. One additional positive arginine residue is created at thejunction site between the first and second cassette, together with aserine. Longer pEXP5CT/(POLY)^(n)-gp144 (pEXP5CT/(POLY)^(n)-KZ144)variants were constructed similarly by repeated cycles.

FIG. 2 shows the expression and secretion of POLY-gp144 by Pichiapastoris . An amount of 30 μl supernatant of a P. pastoris X33expression culture [after 1 day (square), 3 days (triangle) and 4 days(circle)] is added to 270 μl chloroform-permeabilized P. aeruginosaPAO1p cells. The buffer conditions were the optimal enzymatic conditionsof POLY-gp144 (KH₂PO₄/K₂HP0₄) I=120 mM pH 6.2). Subsequently, theoptical density was spectrophotometrically recorded. A drop in opticaldensity indicates the secretion of a muralytic enzyme by P. pastoris .As a negative control, P. pastoris X33 without expression plasmid isincluded (diamond).

FIG. 3 shows in a graphical representation the antibacterial activity ofthe unmodified phiKZgp144 and ELgp188 endolysins, of the modifiedvariants POLY-gp 144 and POLY-gp188 comprising a peptide stretchcomprising 9 positively charged amino acid residues and of the modifiedvariants (POLY)²-gp144 and (POLY)²-gp188 comprising a peptide stretchcomprising 18 positively charged amino acid residues on Pseudomonasaeruginosa PAO1p cells. The error bars render the standard deviations ofthe mean.

FIG. 4 shows a picture of a Coomassie-stained SDS-PAGE showing theresults of the expression and purification of the unmodified endolysinPSP3gp10 and its modified endolysin variant PKPSP3gp10 . The lane LMWpertains to a size marker (LMW ladder). The following three lanespertain to protein fractions of the purified protein in Elution Buffer(20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 500 mM imidazole) after Ni²⁺affinity chromatography. The lane FT pertains to the flow through andthe lane W to waste fractions. Only minor secondary bands are visible inthe purified protein fractions, indicating the high purity of therecombinant proteins (>90%).

FIGS. 5A to D show in a graphic representation the antibacterialactivities of unmodified PSP3gp10 and the modified PKPSP3gp10 indifferent compositions on several exponential growing Gram-negativebacteria after an incubation at room temperature and without shaking.Each species of Gram-negative bacteria was incubated for 30 minutes witha composition comprising 0.5 mM EDTA but no endolysin, with acomposition comprising 1.315 μM unmodified PSP3gp10 but no EDTA, with acomposition comprising 1.315 μM modified PKPSP3gp10 but no EDTA, with acomposition comprising 1.315 μM unmodified PSP3gp10 and 0.5 mM EDTA andwith a composition comprising 1.315 μM modified PKPSP3gp10 and 0.5 mMEDTA. In FIG. 5A the antibacterial activity on P. aeruginosa PAO1p cellsis represented, in FIG. 5B the antibacterial activity on P. aeruginosaBr667 cells, in FIG. 5C he antibacterial activity on E. coli WK 6 cellsand in FIG. 5D the antibacterial activity on Salmonella typhimuriumcells. “Δ” gives the difference of activity between the respectivePSP3gp10 and PKPSP3gp10 samples. The error bars render the standarddeviations of the mean.

FIG. 6 shows a picture of a Coomassie-stained SDS-PAGE showing theresults of the expression and purification of the unmodified endolysinP2gp09 and its modified endolysin variant PKP2gp09 . The lane LMWpertains to a size marker (LMW ladder). The following three lanespertain to protein fractions of the purified protein in Elution Buffer(20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 500 mM imidazole) after Ni²⁺affinity chromatography. The lane FT pertains to the flow through andthe lane W to waste fractions. Only minor secondary bands are visible inthe purified protein fractions, indicating the high purity of therecombinant protein (>95%).

FIGS. 7A to F show in a graphic representation the antibacterialactivities of unmodified P2gp09 and the modified PKP2gp09 in differentcompositions on several exponential growing Gram-negative bacteria afteran incubation at room temperature and without shaking. Each species ofGram-negative bacteria was incubated for 30 minutes with a compositioncomprising 0.5 mM EDTA but no endolysin, with a composition comprising1.315 μM unmodified P2gp09 but no EDTA, with a composition comprising1.315 μM modified PKP2gp09 but no EDTA, with a composition comprising1.315 μM unmodified P2gp09 and 0.5 mM EDTA and with a compositioncomprising 1.315 μM modified PKP2gp09 and 0.5 mM EDTA. In FIG. 7A theantibacterial activity on P. aeruginosa PAO1p cells is represented, inFIG. 7B the antibacterial activity on P. aeruginosa Br667 cells, in FIG.7 C the antibacterial activity on E. coli WK 6 cells, in FIG. 7D theantibacterial activity on Burkholderia pseudomallei cells, in FIG. 7Ethe antibacterial activity on Pseudomonas putida G1 cells and in FIG. 7Fthe antibacterial activity on Salmonella typhimurium LT2 (SGSC N° 2317)cells. “Δ” gives the difference of activity between the respectiveP2gp09 and PKP2gp09 samples. The error bars render the standarddeviations of the mean.

FIG. 8 shows a picture of a Coomassie-stained SDS-PAGE showing theresults of the expression and purification of the unmodified endolysinOBPgpLYS and its modified endolysin variant PKOBPgpLYS. The lane LMWpertains to a size marker (LMW ladder). The following three lanespertain to protein fractions of the purified protein in Elution Buffer(20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 500 mM imidazole) after Ni²⁺affinity chromatography. The lane FT pertains to the flow through andthe lane W to waste fractions. Only minor secondary bands are visible inthe purified protein fractions, indicating the high purity of therecombinant proteins (>90%).

FIGS. 9A to F show in a graphic representation the antibacterialactivities of different compositions of unmodified OBPgpLYS and themodified PKOBPgpLYS on several exponential growing Gram-negativebacteria after an incubation at room temperature and without shaking.Each species of Gram-negative bacteria was incubated for 30 minutes witha composition comprising 0.5 mM EDTA but no endolysin, with acomposition comprising 1.315 μM unmodified OBPgpLYS but no EDTA, with acomposition comprising 1.315 μM modified PKOBPgpLYS but no EDTA, with acomposition comprising 1.315 μM unmodified OBPgpLYS and 0.5 mM EDTA andwith a composition comprising 1.315 μM modified KOBPgpLYS and 0.5 mMEDTA. In FIG. 9A the antibacterial activity on Escherichia coli WK6cells is represented, in FIG. 9B the antibacterial activity onSalmonella typhimurium LT2 (SGSC N° 2317) cells, in FIG. 9C theantibacterial activity on Pseudomonas aeruginosa PAO1p cells, in FIG. 9Dthe antibacterial activity on Pseudomonas aeruginosa Br667 cells, inFIG. 9E the antibacterial activity on Pseudomonas putida G1 cells and inFIG. 9F the antibacterial activity on Burkholderia pseudomallei cells.“Δ” gives the difference of activity between the respective OBPgpLYS andPKOBPgpLYS samples. The error bars render the standard deviations of themean.

The term “protein” as used herein refers synonymously to the term“polypeptide”. The term “protein” as used herein refers to a linearpolymer of amino acid residues linked by peptide bonds in a specificsequence. The amino-acid residues of a protein may be modified by e.g.

covalent attachments of various groups such as carbohydrates andphosphate. Other substances may be more loosely associated with thepolypeptide chains, such as heme or lipid, giving rise to the conjugatedproteins which are also comprised by the term “protein” as used herein.There are various ways in which the polypeptide chains fold have beenelucidated, in particular with regard to the presence of alpha helicesand beta-pleated sheets. The term “protein” as used herein refers to allfour classes of proteins being all-alpha, all-beta, alpha/beta and alphaplus beta.

The term “fusion protein” as used herein refers to an expression productresulting from the fusion of two nucleic acid sequences. Such a proteinmay be produced, e.g., in recombinant DNA expression systems. Moreover,the term “fusion protein” as used herein refers to a fusion of a firstamino acid sequence, in particular an endolysin, autolysin and/or otherpeptidoglycan hydrolase, with a second or further amino acid sequence.The second or further amino acid sequence is preferably a peptidestretch, in particular a cationic and/or polycationic peptide.Preferably, said second and/or further amino acid sequence is foreign toand not substantially homologous with any domain of the first amino acidsequence.

The term “modified endolysin variant” is used herein synonymously withthe term “endolysin variant”. Both terms refer to a fusion proteincomprising an endolysin and a peptide stretch, in particular a cationicand/or polycationic peptide.

The term “peptide stretch” as used herein refers to any kind of peptidelinked to a protein such as an endolysin, autolysin and/or peptidoglycanhydrolase. In particular the term “peptide stretch” as used hereinrefers to a cationic peptide and/or a polycationic peptide. However, apeptide stretch in the meaning of the present invention does not referto His-tags, Strep-tags, Avi-tags, Myc-tags, Gst-tags, JS-tags,cystein-tags, FLAG-tags or other tags known in the art, thioredoxin ormaltose binding proteins (MBP). The term “tag” in contrast to the term“peptide stretch” as used herein refers to a peptide which can be usefulto facilitate expression and/or affinity purification of a polypeptide,to immobilize a polypeptide to a surface or to serve as a marker or alabel moiety for detection of a polypeptide e.g. by antibody binding indifferent ELISA assay formats as long as the function making the taguseful for one of the above listed facilitation is not caused by thepositively charge of said peptide. However, the His-tag may, dependingon the respective pH also be positively charged, but is used as affinitypurification tool as it binds to immobilized divalent cations and is notused as a peptide stretch according to the present invention.

The term “peptide” as used herein refers to short polypeptidesconsisting of from about 2 to about 100 amino acid residues, morepreferably from about 4 to about 50 amino acid residues, more preferablyto about 5 to 30 amino acid residues, wherein the amino group of oneamino acid residue is linked to the carboxyl group of another amino acidresidue by a peptide bond. A peptide may have a specific function. Apeptide can be a naturally occurring peptide or a synthetically designedand produced peptide. The peptide can be, for example, derived orremoved from a native protein by enzymatic or chemical cleavage, or canbe prepared using conventional peptide synthesis techniques (e.g., solidphase synthesis) or molecular biology techniques (see Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989)). Preferred synthetically producedpeptides are e.g. cationic or polycationic peptides.

As used herein, the term “cationic peptide” refers to a peptide havingpositively charged amino acid residues. Preferably a cationic peptidehas a pKa-value of 9.0 or greater. Typically, at least four of the aminoacid residues of the cationic peptide can be positively charged, forexample, lysine or arginine. “Positively charged” refers to the sidechains of the amino acid residues which have a net positive charge atabout physiological conditions. The term “cationic peptide” as usedherein refers also to polycationic peptides.

The term “polycationic peptide” as used herein refers to a syntheticallydesigned and produced peptide composed of mostly positively chargedamino acid residues, in particular lysine, arginine and/or histidineresidues, more preferably lysine and/or arginine residues. A peptide iscomposed of mostly positively charged amino acid residues if at leastabout 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or about 100% of theamino acid residues are positively charged amino acid residues, inparticular lysine and/or arginine residues. The amino acid residuesbeing not positively charged amino acid residues can be neutrallycharged amino acid residues and/or negatively charged amino acidresidues and/or hydrophobic amino acid residues. Preferably the aminoacid residues being not positively charged amino acid residues areneutrally charged amino acid residues, in particular serine and/orglycine.

The term “endolysin” as used herein refers to an enzyme which issuitable to hydrolyse bacterial cell walls. “Endolysins” comprise of atleast one “enzymatically active domain” (EAD) having at least one of thefollowing activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase(amidase), N-acetyl-muramidase, N-acetyl-glucosaminidase (lysozyme) ortransglycosylases. In addition, the endolysins may contain also regionswhich are enzymatically inactive, and bind to the cell wall of the hostbacteria, the so-called CBDs (cell wall binding domains). The endolysinmay contain one, two or more CBDs. However, the term “endolysin” as usedherein refers also to enzymes having at least one EAD but no CBDs.Generally, the cell wall binding domain is able to bind differentcomponents on the surface of bacteria. Preferably, the cell wall bindingdomain is a peptidoglycan binding domain and binds to the bacteria'speptidoglycan.

The term “cell wall” as used herein refers to all components that formthe outer cell enclosure of the Gram-negative bacteria and thusguarantee their integrity. In particular, the term “cell wall” as usedherein refers to peptidoglycan, the outer membrane of the Gram-negativebacteria with the lipopolysaccharide, the bacterial cell membrane, butalso to additional layers deposited on the peptidoglycan as e.g.capsules, outer protein layers or slimes.

The term “autolysins” as used herein refers to enzymes related toendolysins but encoded by bacteria and involved in e.g. cell divisionand cell wall metabolism. An overview of autolysins can be found in“Bacterial peptidoglycan (murein) hydrolases. Vollmer W, Joris B,Charlier P, Foster S. FEMS Microbiol Rev. 2008 March; 32(2):259-86”.

The term “EAD” as used herein refers to the enzymatically active domainof an endolysin. The EAD is responsible for hydrolysing bacterialpeptidoglycans. It exhibits at least one enzymatic activity of anendolysin. The EAD can also be composed of more than one enzymaticallyactive module. The term “EAD” is used herein synonymously with the term“catalytic domain”.

The term “deletion” as used herein refers to the removal of 1, 2, 3, 4,5 or more amino acid residues from the respective starting sequence.

The term “insertion” or “addition” as used herein refers to theinsertion or addition of 1, 2, 3, 4, 5 or more amino acid residues tothe respective starting sequence.

The term “substitution” as used herein refers to the exchange of anamino acid residue located at a certain position for a different one.

The present invention relates to improved antibacterial agents againstGram-negative bacteria, in case modified endolysin variants, comprisingan endolysin fused to a peptide with lipopolysachharide (LPS) or ingeneral membrane disrupting activity. LPS is a major component of theouter membrane of Gram-negative bacteria. It increases the negativecharge of the cell membrane and protects the membrane from certain kindsof chemical attack. To a certain degree said LPS protects the membraneof Gram-negative bacteria also from endolysins added from outside of thebacteria. However, the LPS can be disrupted by peptide stretches havinga LPS disrupting activity as e.g. positively charged peptides. Moreover,said peptide stretches may be involved in the outer membrane proteintransport mechanism, a destabilisation of structural outer membraneproteins and/or in lipid-dependent destabilisation. The inventors of thepresent invention have surprisingly found, that a peptide stretch havingLPS disrupting activity or in general membrane disrupting activitypromotes the passage of an endolysin fused to said peptide stretchthrough the outer membrane of Gram-negative bacteria. After the promotedpass of the endolysin through the outer membrane of Gram-negativebacteria, the cell wall of the Gram-negative bacterium can be moreeasily be disrupted or desintegrated by the endolysin due to degradationof the peptidoglycan layer followed by osmotic lysis when the internalcell pressure of the bacterium cannot longer be resisted.

Thus, the present invention refers to fusion proteins composed of anendolysin having the activity of degrading the cell wall ofGram-negative bacteria and a peptide stretch with membrane disruptingactivity, wherein said peptide stretch is fused to the enzyme at the N-and/or C-terminus. Said fusion proteins according to the presentinvention are also called modified endolysin variants or simplyendolysin variants or modified endolysins.

The endolysin part of the modified endolysin variant is preferablyencoded by bacteriophages specific for Gram-negative bacteria such asGram-negative bacteria of bacterial groups, families, genera or speciescomprising strains pathogenic for humans or animals likeEnterobacteriaceae (Escherichia, especially E. coli, Salmonella,Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella,especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia,Yersinia), Pseudomonadaceae (Pseudomonas, especially P. aeruginosa,Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas),Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella,Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella,Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema andBorrelia), Leptospiraceae, Campylobacter, Helicobacter, Spirillum,Streptobacillus, Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella,Porphyromonas), Acinetobacter, especially A. baumanii.

Moreover, the endolysin has preferably cell wall degrading activityagainst Gram-negative bacteria of bacterial groups, families, genera orspecies comprising strains pathogenic for humans or animals likeEnterobacteriaceae (Escherichia, especially E. coli, Salmonella,Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella,especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia,Yersinia), Pseudomonadaceae (Pseudomonas, especially P. aeruginosa,Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas),Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella,Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella,Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema andBorrelia), Leptospiraceae, Campylobacter, Helicobacter, Spirillum,Streptobacillus, Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella,Porphyromonas), Acinetobacter, especially A. baumanii.

Preferably, the endolysin part derives from a phage or a wild typeendolysin as depicted in the following table:

phage publication Wild type endolysin predicted function of theendolysin φV10 Perry, L. L. and Applegate, B. M. PhiV10p30 chitinaseFELS-1 McClelland, M. and Wilson, R. K. STM0907.Fels0 chitinase ε15Kropinksi, A. M. and McConnel, M. R. epsilon15p25 chitinase YUACeyssens. P. (Laboratory for Gene YuA20 lytic transglycosylase (C)/1transmembranair technology) domain (N) B3 Braid, M. D. and Kitts, C. L.ORF23 lytic transglycosylase (C)/2 transmembranair domains (N) BCEPμSummer, E. J. and Young, R. BcepMu22 lytic transglycosylase (M)/1transmembranair domain (N) F116 Byrne, M. and Kropinski, A. M. F116p62muraminidase (T4-like) FELS-2 McClelland, M. and Wilson, R. K.STM2715.S.Fels2 muraminidase (T4-like) ES18 Casjens, S. R. and Hendrix,R. W. gp76 muraminidase (T4-like) SETP3 De Lappe, N and Cormican, M.SPSV3_gp23 muraminidase (T4-like) φECO32 Savalia, D and Severinov, Kphi32_17 muraminidase (T4-like) HK022 Juhala, R and Hendrix, R. W.HK022p54 muraminidase (lambdalike) HK97 Juhala, R and Hendrix, R. W.HK97p58 muraminidase (lambdalike) HK620 Clark, A. J. and Dhillon, T. S.HK620p36 muraminidase (lambdalike) E1 Pickard, D. and Dougan, G VIP0007muraminidase (lambdalike) SF6 Casjens, S and Clark, A. J. Sf6p62muraminidase (lambdalike) SFV Allison, G. E. and Verma, N. K. R (SfVp40)muraminidase (lambdalike) BCEPC6B Summer, E J and Young, R. gp22muraminidase (lambdalike) BCEPNAZGUL Summer, E J and Young, R. Nazgul38muraminidase (lambdalike) P2 Christie, G. E. and Calender, R. K (P2p09)muraminidase (lambdalike) Wφ Christie, G. E. and Esposito, D. K (Wphi09)muraminidase (lambdalike) RV5 Kropinski, A. M. and Johnson rv5_gp085muraminidase (lambdalike) JS98 Zuber, S and Denou, E. EpJS98_gp116muraminidase (T4-like) 13A Savalia, D and Molineux, I. gp3.5muramoyl-L-alanine amidase BA14 Savalia, D and Molineux, I. gp3.5muramoyl-L-alanine amidase ECODS1 Savalia, D and Molineux, I. gp3.5muramoyl-L-alanine amidase K1F Scholl, D and Merril, C CKV1F_gp16muramoyl-L-alanine amidase T3 Pajunen, M. I. and Mollineux, I. J. T3p18muramoyl-L-alanine amidase GH-1 Kropinski, A. M. and Kovalyova, I. V.gh-1p12 muramoyl-L-alanine amidase K11 Molineux, I. and Savalia, D.gp3.5 muramoyl-L-alanine amidase BIP-1 Liu, M and Miller, J. F. bip-1p02lysozyme (N)/PG-binding domain (C) BMP-1 Liu, M and Miller, J. F.bmp-1pO2 lysozyme (N)/PG-binding domain (C) BPP-1 Liu, M and Miller, J.F. bpp2 lysozyme (N)/PG-binding domain (C) φCTX Nakayama, K and Hayashi,T. ORF12 PG-binding domain (N)/muramidase (C) BCEP43 Summer, E J andYoung, R. Bcep43-27 PG-binding domain (N)/muramidase (C) BCEP781 Summer,E J and Young, R. Bcep781-27 PG-binding domain (N)/muramidase (C) BCEP1Summer, E J and Young, R. Bcep1-28 PG-binding domain (N)/muramidase (C)BCEPNY3 Summer, E J and Young, R. BcepNY3gene26 PG-binding domain(N)/muramidase (C) φE12-2 DeShazer, D and Nierman, W. C. gp45 PG-bindingdomain (N)/muramidase (C) φ52237 DeShazer, D and Nierman, W. C. gp28PG-binding domain (N)/muramidase (C) φP27 Recktenwald, J and Schmidt, H.P27p30 endopeptidase RB49 Monod, C and Krisch, H. M. RB49p102endopeptidase φ1 Arbiol, C. and Comeau, A. M. phi1-p102 endopeptidase T5Pankova, N. V. and Ksenzenko, V. N. lys (T5.040) endopeptidase 201phi2-1Thomas et al., 2008 PG-binding domain (N)/unknown catalytic domain (C)Aeh1 Monod, C and Krisch, H. M. Aeh1p339 muraminidase (T4-like) YYZ-2008Kropinski, A. M. YYZgp45 muraminidase (lambda-like)

Also preferred is the endolysin part deriving from endolysins of thePseudomonas aeruginosa phages ΦKZ and EL, of the Pseudomonas putidaphage OBP, of the phage LUZ24, or from T4 lysozyme, gp61 muramidase andPSP3 endolysin.

More preferably, the endolysin part is selected from the groupconsisting of phiKZgp144 according to SEQ ID NO:1, ELgp188 according toSEQ ID NO:2, Salmonella endolysin according to SEQ ID NO:3,Enterobacteria phage T4 endolysin according to SEQ ID NO:4,Acinetobacter baumanii endolysin according to SEQ ID NO:5, E. coli PhageK1F endolysin according to SEQ ID NO:6, OBPgpLYS according to SEQ ID NO:7, PSP3 Salmonella endolysin (PSP3gp10) according to SEQ ID NO: 8 and E.coli Phage P2 endolysin (P2gp09) according to SEQ ID NO: 9.

In another preferred embodiment of the present invention the endolysinsor the modified endolysin variants according to the present inventioncomprise modifications and/or alterations of the amino acid sequences.Such alterations and/or modifications may comprise mutations such asdeletions, insertions and additions, substitutions or combinationsthereof and/or chemical changes of the amino acid residues, e.g.biotinylation, acetylation, PEGylation, chemical changes of the amino-,SH- or carboxyl-groups. Said modified and/or altered endolysins exhibitthe lytic activity of the respective wild type endolysin. However, saidactivity can be higher or lower as the activity of the respective wildtype endolysin. Said activity can be about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200%of the activity of the respective wild-type endolysin or even more. Theactivity can be measured by assays well known in the art by a personskilled in the art as e.g. the plate lysis assay or the liquid lysisassay which are e.g. described in (Briers et al., J. Biochem. BiophysMethods 70: 531-533, (2007)).

In one aspect of the invention the peptide with membrane and/or LPSdisrupting activity comprises a positively charged peptide, whichcomprises one or more of the positively charged amino acids beinglysine, arginine and/or histidine. Preferably, more than 80%, preferablymore than 90%, preferably 100% of the amino acids in said peptide arepositively charged amino acids. Advantageously, the cationic peptide isfused at the N-terminal and/or the C-terminal end of the endolysinvariants, thus enhancing the cationicity of the latter proteins. Inanother embodiment of the invention, the cationic peptide fused to theendolysin is at least 5, more preferably at least 9 amino acids long.

In a preferred embodiment the endolysin variant comprises an endolysinand a peptide fused thereto said peptide comprising about 3 to about 50,more preferably about 5 to about 20, for instance about 5 to about 15amino acid residues and at least 20, 30, 40, 50, 60 or 70%, morepreferably at least 80%, for instance at least 90% of the said aminoacid residues are either arginine or lysine residues. In anotherpreferred embodiment the endolysin variant comprises an endolysin and apeptide fused thereto said peptide comprising about 3 to about 50, morepreferably about 5 to about 20, for instance about 5 to about 15 aminoacid residues and said amino acid residues are either arginine or lysineresidues.

Preferably, the peptide stretch of the modified endolysin variant isfused to the N-terminus and/or to the C-terminus of the endolysin. In aparticular preferred embodiment said peptide stretch is only fused tothe N-terminus of the endolysin. However, also preferred are modifiedendolysin variants having a peptide stretch both on the N-terminus andon the C-terminus. Said peptide stretches on the N-terminus and on theC-terminus can be the same or distinct peptide stretches.

The peptide stretch of the modified endolysin variant according to thepresent invention is preferably covalently bound to the enzyme.Preferably, said peptide stretch consists of at least 5, more preferablyat least of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or at least 100 amino acid residues. Especiallypreferred is a peptide stretch comprising about 5 to about 100 aminoacid residues, about 5 to about 50 or about 5 to about 30 amino acidresidues. More preferred is a peptide stretch comprising about 6 toabout 42 amino acid residues, about 6 to about 39 amino acid residues,about 6 to about 38 amino acid residues, about 6 to about 31 amino acidresidues, about 6 to about 25 amino acid residues, about 6 to about 24amino acid residues, about 6 to about 22 amino acid residues, about 6 toabout 21 amino acid residues, about 6 to about 20 amino acid residues,about 6 to about 19 amino acid residues, about 6 to about 16 amino acidresidues, about 6 to about 14 amino acid residues, about 6 to about 12amino acid residues, about 6 to about 10 amino acid residues or about 6to about 9 amino acid residues.

In one aspect of the present invention the fused peptide stretch is acationic and/or polycationic peptide, which comprises one or more of thepositively charged amino acid residues of lysine, arginine and/orhistidine, in particular of lysine and/or arginine. Preferably, morethan about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 99% of theamino acid residues in said peptide stretch are positively charged aminoacid residues, in particular lysine and/or arginine residues. Especiallypreferred are peptide stretches consisting of about 100% positivelycharged amino acid residues, in particular arginine and/or lysineresidues, wherein preferably about 60% to about 70% of said positivelycharged amino acid residues are lysine residues and about 30% to about40% of said positively charged amino acid residues are arginineresidues. More preferred is a peptide stretch consisting of about 100%positively charged amino acid residues, in particular arginine and/orlysine residues, wherein preferably about 64% to about 68% of saidpositively charged amino acid residues are lysine and about 32% to about36% of said positively charged amino acid residues are arginine. Peptidestretches consisting of either only arginine or only lysine are alsopreferred.

Especially preferred are cationic and/or polycationic peptide stretchescomprising at least one motive according to SEQ ID NO: 10 (KRKKRK). Inparticular cationic peptide stretches comprising at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 motives according to SEQ IDNO: 10 (KRKKRK) are preferred. More preferred are cationic peptidestretches comprising at least one KRK motive (lys-arg-lys), preferableat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 KRK motives.

In another preferred embodiment of the present invention the cationicpeptide stretch comprises beside the positively charged amino acidresidues, in particular lysine and/or arginine residues, neutrallycharged amino acid residues, in particular glycine and/or serineresidues. Preferred are cationic peptide stretches consisting of about70% to about 100%, or about 80% to about 95%, or about 85% to about 90%positively charged amino acid residues, in particular lysine, arginineand/or histidine residues, more preferably lysine and/or arginineresidues and of about 0% to about 30%, or about 5% to about 20%, orabout 10% to about 20% neutrally charged amino acid residues, inparticular glycine and/or serine residues. Preferred are polypeptidestretches consisting of about 4% to about 8% serine residues, of about33% to about 36% arginine residues and of about 56% to about 63% lysineresidues. Especially preferred are polypeptide stretches comprising atleast one motive according to SEQ ID NO: 32 (KRXKR), wherein X is anyother amino acid than lysine, arginine and histidine. Especiallypreferred are polypeptide stretches comprising at least one motiveaccording to SEQ ID NO: 33 (KRSKR). More preferred are cationicstretches comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or about 20 motives according to SEQ ID NO:32 (KRXKR) or SEQ ID NO: 33 (KRSKR).

Also preferred are polypeptide stretches consisting of about 9 to about16% glycine residues, of about 4 to about 11% serine residues, of about26 to about 32% arginine residues and of about 47 to about 55% lysineresidues. Especially preferred are polypeptide stretches comprising atleast one motive according to SEQ ID NO: 34 (KRGSG). More preferred arecationic stretches comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 motives according to SEQID NO: 34 (KRGSG).

In another preferred embodiment of the present invention the cationicpeptide stretch comprises beside the positively charged amino acidresidues, in particular lysine and/or arginine residues, hydrophobicamino acid residues, in particular valine, isoleucine, leucine,methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine,histidine, threonin, serine, proline and glycine residues, morepreferably alanine, valine, leucine, isoleucine, phenylalanine, and/ortryptophan residues. Preferred are cationic peptide stretches consistingof about 70% to about 100%, or about 80% to about 95%, or about 85% toabout 90% positively charged amino acid residues, in particular lysineand/or arginine residues and of about 0% to about 30%, or about 5% toabout 20%, or about 10% to about 20% hydrophobic amino acid residues,valine, isoleucine, leucine, methionine, phenylalanine, tryptophan,cysteine, alanine, tyrosine, histidine, threonin, serine, proline andglycine residues, more preferably alanine, valine, leucine, isoleucine,phenylalanine, and/or tryptophan residues.

Especially preferred are peptide stretches selected from the groupconsisting of the following sequences:

peptide stretch length SEQ ID NO: KRKKRK  6 SEQ ID NO: 10 KRKKRKKRK  9SEQ ID NO: 11 RRRRRRRRR  9 SEQ ID NO: 12 KKKKKKKK  8 SEQ ID NO: 13KRKKRKKRKK 10 SEQ ID NO: 14 KRKKRKKRKKRK 12 SEQ ID NO: 15 KRKKRKKRKKRKKR14 SEQ ID NO: 16 KKKKKKKKKKKKKKKK 16 SEQ ID NO: 17 KRKKRKKRKKRKKRKKRKK19 SEQ ID NO: 18 RRRRRRRRRRRRRRRRRRR 19 SEQ ID NO: 19KKKKKKKKKKKKKKKKKKK 19 SEQ ID NO: 20 KRKKRKKRKRSKRKKRKKRK 20SEQ ID NO: 21 KRKKRKKRKRSKRKKRKKRKK 21 SEQ ID NO: 22KRKKRKKRKKRKKRKKRKKRK 21 SEQ ID NO: 23 KRKKRKKRKRGSGKRKKRKKRK 22SEQ ID NO: 24 KRKKRKKRKRGSGSGKRKKRKKRK 24 SEQ ID NO: 25KRKKRKKRKKRKKRKKRKKRKKRKK 25 SEQ ID NO: 26KRKKRKKRKRSKRKKRKKRKRSKRKKRKKRK 31 SEQ ID NO: 27KRKKRKKRKRGSGSGKRKKRKKRKGSGSGKRKKRKKRK 38 SEQ ID NO: 28KRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRK 39 SEQ ID NO: 29KRKKRKKRKRSKRKKRKKRKRSKRKKRKKRKRSKRKKRKKRK 42 SEQ ID NO: 30

Preferably, the peptide stretch is no tag such as a His-tag, Strep-tag,Avi-tag, Myc-tag, Gst-tag, JS-tag, cystein-tag, FLAG-tag or other tagsknown in the art and no thioredoxin or maltose binding proteins (MBP).However, the peptide stretch and/or the modified endolysin variantaccording to the present invention may comprise in addition such tag ortags.

Preferably, the peptide stretch has the function to lead the modifiedendolysin variant according to the present invention through the outermembrane of Gram-negative bacteria but has no or only low activity whenadministered without being fused to the enzyme. The function to lead themodified endolysin variant through the outer membrane of Gram-negativebacteria is caused by the potential of the outer membrane or LPSdisrupting activity of said peptide stretch.

Especially preferred are modified endolysin variants selected from thegroup consisting of the following modified endolysin variants:

SEQ ID NO: Peptide stretch Modified endolysin (modified Endolysin(N-terminal unless variant endolysin variant) part otherwise indicated)POLY-gp144 SEQ ID NO: 35 SEQ ID NO: 1 SEQ ID NO: 11 (POLY)²-gp144 SEQ IDNO: 36 SEQ ID NO: 1 SEQ ID NO: 21 (POLY)³-gp144 SEQ ID NO: 37 SEQ ID NO:1 SEQ ID NO: 27 (POLY)⁴-gp144 SEQ ID NO: 38 SEQ ID NO: 1 SEQ ID NO: 30POLY-gp188 SEQ ID NO: 39 SEQ ID NO: 2 SEQ ID NO: 11 (POLY)²-gp188 SEQ IDNO: 40 SEQ ID NO: 2 SEQ ID NO: 21 (POLY)³-gp188 SEQ ID NO: 41 SEQ ID NO:2 SEQ ID NO: 27 (POLY)⁴-gp188 SEQ ID NO: 42 SEQ ID NO: 2 SEQ ID NO: 30pKKZ144pET32b SEQ ID NO: 43 SEQ ID NO: 1 SEQ ID NO: 14 KRK_6_pET32b SEQID NO: 44 SEQ ID NO: 1 SEQ ID NO: 10 KRK_12_pET32b SEQ ID NO: 45 SEQ IDNO: 1 SEQ ID NO: 15 KRK_14_pET32b SEQ ID NO: 46 SEQ ID NO: 1 SEQ ID NO:16 R9_pET32b SEQ ID NO: 47 SEQ ID NO: 1 SEQ ID NO: 12 K8_pET32b SEQ IDNO: 48 SEQ ID NO: 1 SEQ ID NO: 13 pK2KZ144_pET32b_mod3 SEQ ID NO: 49 SEQID NO: 1 SEQ ID NO: 28 PKPSP3gp10 SEQ ID NO: 53 SEQ ID NO: 8 SEQ ID NO:11 PKP2gp09 SEQ ID NO: 57 SEQ ID NO: 9 SEQ ID NO: 11 PKOBPgpLYS SEQ IDNO: 61 SEQ ID NO: 7 SEQ ID NO: 11 pK2KZ144pET32b SEQ ID NO: 62 SEQ IDNO: 1 SEQ ID NO: 22 pK3KZ144pET32b SEQ ID NO: 63 SEQ ID NO: 1 SEQ ID NO:27 pK4KZ144pET32b SEQ ID NO: 64 SEQ ID NO: 1 SEQ ID NO: 30 KRK_19_pET32bSEQ ID NO: 66 SEQ ID NO: 1 SEQ ID NO: 18 KRK_21_pET32b SEQ ID NO: 67 SEQID NO: 1 SEQ ID NO: 23 KRK_25_pET32b SEQ ID NO: 68 SEQ ID NO: 1 SEQ IDNO: 26 KRK_39_pET32b SEQ ID NO: 69 SEQ ID NO: 1 SEQ ID NO: 29 K19_pET32bSEQ ID NO: 70 SEQ ID NO: 1 SEQ ID NO: 20 K16_pET32b SEQ ID NO: 71 SEQ IDNO: 1 SEQ ID NO: 17 pKKZ-144_K2_pET32b SEQ ID NO: 72 SEQ ID NO: 1N-terminal: SEQ ID NO: 11 C-teiminal: SEQ ID NO: 21 pK2KZ144_pET32b_mod1SEQ ID NO: 73 SEQ ID NO: 1 SEQ ID NO: 24 pK2KZ144_pET32b_mod2 SEQ ID NO:74 SEQ ID NO: 1 SEQ ID NO: 25 smi01_KRK9 SEQ ID NO: 75 SEQ ID NO: 1 SEQID NO: 11 smi02_KRK9 SEQ ID NO: 76 SEQ ID NO: 1 SEQ ID NO: 11 smi03_KRK9SEQ ID NO: 77 SEQ ID NO: 1 SEQ ID NO: 11 smi04_KRK9 SEQ ID NO: 78 SEQ IDNO: 1 SEQ ID NO: 11

The modified endolysin variants according to the present invention, andthus in particular the especially preferred modified endolysin variantsaccording to SEQ ID NO: 35 to 49, 53, 57, 61 to 64 and 66 to 78, mayadditional comprise a tag e.g. for purification. Preferred is aHis₆-tag, preferably at the C-terminus of the modified endolysinvariant. Said tag can be linked to the modified endolysin variant byadditional amino acid residues e.g. due to cloning reasons. Preferablysaid tag can be linked to the modified endolysin variant by at least 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid residues. In apreferred embodiment the modified endolysin variant comprises a His₆-tagat its C-terminus linked to the modified endolysin variant by theadditional amino acid residues lysine and glycine (Lys-Gly) or leucineand glutamic acid (Leu-Glu).

In particular, the modified endolysin variants as used in the examplesas described below are preferred. The modified endolysin variantsaccording to SEQ ID NO: 35 to 42, 53, 57 and 61 as used in the examplescomprise a His₆-tag at the C-terminus linked to the respective modifiedendolysin variant by the additional amino acid residues lysine andglycine (Lys-Gly). The modified endolysin variants according to SEQ IDNO: 43 to 49 and 75 as used in the examples comprise a His₆-tag at theC-terminus linked to the respective modified endolysin variant by theadditional amino acid residues leucine and glutamic acid (Leu-Glu).

Fusion proteins are constructed by linking at least two nucleic acidsequences using standard cloning techniques as described e.g. bySambrook et al. 2001, Molecular Cloning: A Laboratory Manual. Such aprotein may be produced, e.g., in recombinant DNA expression systems.Such fusion proteins according to the present invention can be obtainedby fusing the nucleic acids for endolysin and the respective peptidestretch.

As some fusion proteins may either be toxic upon expression in bacteria,or not homogenous due to protein degradation, the strategy might be toexpress these fusion proteins fused or linked to other additionalproteins. Example for these other additional protein is Thioredoxin,which was shown to mediate expression of toxic antimicrobial peptides inE. coli (TrxA mediating fusion expression of antimicrobial peptide CM4from multiple joined genes in Escherichia coli . Zhou L, Zhao Z, Li B,Cai Y, Zhang S. Protein Expr Purif. 2009 April; 64(2):225-230).

For antimicrobial function of the fusion proteins it may be necessary toremove the additional fusion protein by proteolytic cleavage.Commercially available kits like the pET32 expression system (Novagen),may need to modify e.g. the N-terminus of the fusion depending on theprotease used, like from MGS to AMGS (SEQ ID NO: 31), were the remainingalanine residue results from an introduced Enterokinase cleavage site.

In another preferred embodiment of the present invention the peptidestretches of the modified endolysin variant according to the presentinvention comprise modifications and/or alterations of the amino acidsequences. Such alterations and/or modifications may comprise mutationssuch as deletions, insertions and additions, substitutions orcombinations thereof and/or chemical changes of the amino acid residues,e.g. biotinylation, acetylation, PEGylation, chemical changes of theamino-, SH- or carboxyl-groups.

The present invention further relates to an isolated nucleic acidmolecule encoding the modified endolysin variant according to thepresent invention. The present invention further relates to a vectorcomprising the nucleic acid molecule according to the present invention.Said vector may provide for the constitutive or inducible expression ofsaid modified endolysin variant according to the present invention.

The invention also relates to a method for obtaining said modifiedendolysin variants from a micro-organism, such as a genetically modifiedsuitable host cell which expresses said modified endolysin variants.Said host cell may be a micro-organism such as bacteria or yeast orfungi or an animal cell as e.g. a mammalian cell, in particular a humancell. In one embodiment of the present invention the yeast cell is aPichia pastoris cell. The host may be selected due to merebiotechnological reasons, e.g. yield, solubility, costs, etc. but may bealso selected from a medical point of view, e.g. a non-pathologicalbacteria or yeast, human cells.

Another aspect of the present invention is related to a method forgenetically transforming a suitable host cell in order to obtain theexpression of the modified endolysin variants according to the inventionwherein the host cell is genetically modified by the introduction of agenetic material encoding said modified endolysin variants into the hostcell and obtain their translation and expression by genetic engineeringmethods well known by a person skilled in the art.

In a further aspect the present invention relates to a composition,preferably a pharmaceutical composition, comprising a modified endolysinvariant according to the present invention and/or a host transformedwith a nucleic acid molecule or a vector comprising a nucleotidesequence encoding a modified endolysin variant according to the presentinvention.

In a preferred embodiment of the present invention the compositioncomprises additionally agents permeabilizing the outer membrane ofGram-negative bacteria such metal chelators as e.g. EDTA, TRIS, lacticacid, lactoferrin, polymyxin, citric acid and/or other substances asdescribed e.g. by Vaara (Agents that increase the permeability of theouter membrane. Vaara M. Microbiol Rev. 1992 September; 56(3):395-441).Also preferred are compositions comprising combinations of the abovementioned permeabilizing agents. Especially preferred is a compositioncomprising about 10 μM to about 100 mM EDTA, more preferably about 50 μMto about 10 mM EDTA, more preferably about 0.5 mM to about 10 mM EDTA,more preferably about 0.5 mM to about 2 mM EDTA, more preferably about0.5 mM to 1 mM EDTA. However, also compositions comprising about 10 μMto about 0.5 mM EDTA are preferred. Also preferred is a compositioncomprising about 0.5 mM to about 2 mM EDTA, more preferably about 1 mMEDTA and additionally about 10 to about 100 mM TRIS.

The present invention also relates to a modified endolysin variantaccording to the present invention and/or a host transformed with anucleic acid comprising a nucleotide sequence encoding a modifiedendolysin variant according to the present invention for use as amedicament.

In a further aspect the present invention relates to the use of amodified endolysin variant according to the present invention and/or ahost transformed with a vector comprising a nucleic acid moleculecomprising a nucleotide sequence encoding a modified endolysin variantaccording to the present invention in the manufacture of a medicamentfor the treatment and/or prevention of a disorder, disease or conditionassociated with pathogenic Gram-negative bacteria. In particular thetreatment and/or prevention of the disorder, disease or condition may becaused by Gram-negative bacteria of bacterial groups, families, generaor species comprising strains pathogenic for humans or animals likeEnterobacteriaceae (Escherichia, especially E. coli, Salmonella,Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella,especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia,Yersinia), Pseudomonadaceae (Pseudomonas, especially P. aeruginosa,Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas),Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella,Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella,Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema andBorrelia), Leptospiraceae, Campylobacter, Helicobacter, Spirillum,Streptobacillus, Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella,Porphyromonas), Acinetobacter, especially A. baumanii . Preferably, saiddisorder, disease or condition may be caused by Pseudomonas, inparticular Pseudomonas aeruginosa and/or Pseudomonas putida,Burkholderia, in particular Burkholderia pseudomallei and/orBurkholderia solanacearum, Salmonella, in particular Salmonellatyphimurium and/or Salmonella Enteritidis, Acinetobacter, in particularAcinetobacter baumannii, Escherichia coli and/or Klebsiella, inparticular Klebsiella pneumoniae.

The present invention further relates to a medicament comprising amodified endolysin variant according to the present invention and/or ahost transformed with a nucleic acid comprising a nucleotide sequenceencoding a modified endolysin variant according to the presentinvention.

In a further aspect the present invention relates to a method oftreating a disorder, disease or condition in a subject in need oftreatment and/or prevention, which method comprises administering tosaid subject an effective amount of a modified endolysin variantaccording to the present invention and/or an effective amount of a hosttransformed with a nucleic acid comprising a nucleotide sequenceencoding a modified endolysin variant according to the present inventionor a composition according to the present invention. The subject may bea human or an animal.

Preferably said method of treatment may be for the treatment and/orprevention of infections caused by Gram-negative bacteria, in particularby the Gram-negative bacteria as listed above. In particular said methodof treatment may be for the treatment and/or prevention of infections ofthe skin, of soft tissues, the respiratory system, the lung, thedigestive tract, the eye, the ear, the teeth, the nasopharynx, themouth, the bones, the vagina, of wounds of bacteraemia and/orendocarditis caused by Gram-negative bacteria, in particular by theGram-negative bacteria as listed above.

The dosage and route of administration used in a method of treatment (orprophylaxis) according to the present invention depends on the specificdisease/site of infection to be treated. The route of administration maybe for example oral, topical, nasopharyngeal, parenteral, inhalational,intravenous, intramuscular, intrathecal, intraspinal, endobronchial,intrapulmonal, intraosseous, intracardial, intraarticular, rectal,vaginal or any other route of administration.

For application of a modified endolysin variant according to the presentinvention and/or an effective amount of a host transformed with anucleic acid comprising a nucleotide sequence encoding a modifiedendolysin variant according to the present invention or a compositionaccording to the present invention to a site of infection (or siteendangered to be infected) a formulation may be used that protects theactive compounds from environmental influences such as proteases,oxidation, immune response etc., until it reaches the site of infection.Therefore, the formulation may be capsule, dragee, pill, powder,suppository, emulsion, suspension, gel, lotion, cream, salve, injectablesolution, syrup, spray, inhalant or any other medical reasonable galenicformulation. Preferably, the galenic formulation may comprise suitablecarriers, stabilizers, flavourings, buffers or other suitable reagents.For example, for topical application the formulation may be a lotion,cream, gel, salve or plaster, for nasopharyngeal application theformulation may be saline solution to be applied via a spray to thenose. For oral administration in case of the treatment and/or preventionof a specific infection site e.g. in the intestine, it can be necessaryto protect a modified endolysin variant according to the presentinvention from the harsh digestive environment of the gastrointestinaltract until the site of infection is reached. Thus, bacteria as carrier,which survive the initial steps of digestion in the stomach and whichsecret later on a modified endolysin variant according to the presentinvention into the intestinal environment can be used.

In a specific embodiment of the present invention the use of a modifiedendolysin variant according to the present invention and/or a hosttransformed with a vector comprising a nucleic acid molecule comprisinga nucleotide sequence encoding a modified endolysin variant according tothe present invention in the manufacture of a medicament for thetreatment and/or prevention of a disorder, disease or condition causedby Pseudomonas, particularly by Pseudomonas aeruginosa in particularintestinal affections, in particular in infants, infections of themeninges, e.g. meningitis haemorrhagica, infections of the middle ear,the skin (Ecthyma gangraenosum), in particular burns, the urinary tract,rhinitis, bacteremic pneumonia, in particular wherein the patient issuffering from cystic fibrosis or hematologic malignancies such asleukemia, or with neutropenia from immunosuppressive therapy,septicemia, in particular because of long-term intravenous or urinarycatheterization, invasive surgical procedures and severe burns,endocarditis, in particular wherein the patient is a intravenous druguser or a patient with complications from open heart surgery, highlydestructive ocular infections, in particular after the use ofcontaminated ophthalmologic solutions or severe facial burns,osteochondritis, in particular as a result of severe trauma or puncturewounds through contaminated clothing.

In another specific embodiment of the present invention the disorder,disease or condition is caused by Burkholderia pseudomallei, inparticular Whitmore's Disease, chronic pneumonia, septicemia, inparticular wherein the patient has a traumatized skin lesion.

In another specific embodiment of the present invention the disorder,disease or condition is caused by Salmonella thyphimurium and Salmonellaenteritidis, in particular acute gastroenteritis and local purulentprocesses, particularly osteomyelitis, endocarditis, cholecystitis andespecially caused by Salmonella thyphimurium meningitis, in particularwherein the patient is less than two years old.

In another specific embodiment of the present invention the disorder,disease or condition is caused by Acinetobacter baumannii, in particularbronchitis, pneumonia, wound infections and septicemia, in particular asa result of intravenous catheterization.

In another specific embodiment of the present invention the disorder,disease or condition is caused by Escherichia coli, in particular extraintestinal infections, particularly appendicitis, purulentcholecystitis, peritonitis, purulent meningitis and infection of theurinary tract, intraintestinal E. coli infections, particularly epidemicenteritis, and infectious disease similar to dysentery, septicemia,enterotoxemia, mastitis and dysentery.

In another specific embodiment of the present invention the disorder,disease or condition is caused by Klebsiella pneumoniae, in particularpneumonia, bacteremia, meningitis and infections of the urinary tract.

Preferably, a modified endolysin variant according to the presentinvention is used for medical treatment, if the infection to be treated(or prevented) is caused by multiresistant bacterial strains, inparticular by strains resistant against one or more of the followingantibiotics: streptomycin, tetracycline, cephalothin, gentamicin,cefotaxime, cephalosporin, ceftazidime or imipenem. Furthermore, amodified endolysin variant according to the present invention can beused in methods of treatment by administering it in combination withconventional antibacterial agents, such as antibiotics, lantibiotics,bacteriocins or endolysins, etc.

The present invention also relates to a pharmaceutical pack comprisingone or more compartments, wherein at least one compartment comprises oneor more modified endolysin variant according to the present inventionand/or one or more hosts transformed with a nucleic acid comprising anucleotide sequence encoding a modified endolysin variant according tothe present invention or a composition according to the presentinvention.

In another aspect the present invention relates to a process ofpreparation of a pharmaceutical composition, said process comprisingadmixing one or more modified endolysin variant according to the presentinvention and/or one or more hosts transformed with a nucleic acidcomprising a nucleotide sequence encoding a modified endolysin variantaccording to the present invention with a pharmaceutically acceptablediluent, excipient or carrier.

In an even further aspect the composition according to the presentinvention is a cosmetic composition. Several bacterial species can causeirritations on environmentally exposed surfaces of the patient's bodysuch as the skin. In order to prevent such irritations or in order toeliminate minor manifestations of said bacterial pathogens, specialcosmetic preparations may be employed, which comprise sufficient amountsof the modified endolysin variant according to the present invention inorder to degrade already existing or freshly settling pathogenicGram-negative bacteria.

In a further aspect the present invention relates to the modifiedendolysin variant according to the present invention for use asdiagnostic means in medicinal, food or feed or environmentaldiagnostics, in particular as a diagnostic means for the diagnostic ofbacteria infection caused in particular by Gram-negative bacteria. Inthis respect the modified endolysin variant according to the presentinvention may be used as a tool to specifically degrade pathogenicbacteria, in particular Gram-negative pathogenic bacteria. Thedegradation of the bacterial cells by the modified endolysin variantaccording to the present invention can be supported by the addition ofdetergents like Triton X-100 or other additives which weaken thebacterial cell envelope like polymyxin B. Specific cell degradation isneeded as an initial step for subsequent specific detection of bacteriausing nucleic acid based methods like PCR, nucleic acid hybridization orNASBA (Nucleic Acid Sequence Based Amplification), immunological methodslike IMS, immunofluorescence or ELISA techniques, or other methodsrelying on the cellular content of the bacterial cells like enzymaticassays using proteins specific for distinct bacterial groups or species(e.g. β-galactosidase for enterobacteria, coagulase for coagulasepositive strains).

In a further aspect the present invention relates to the use of themodified endolysin variant according to the present invention for theremoval, reduction and/or prevention of Gram-negative bacterialcontamination of foodstuff, of food processing equipment, of foodprocessing plants, of surfaces coming into contact with foodstuff suchas shelves and food deposit areas and in all other situations, wherepathogenic, facultative pathogenic or other undesirable bacteria canpotentially infest food material, of medical devices and of all kind ofsurfaces in hospitals and surgeries.

In particular, a modified endolysin variant of the present invention maybe used prophylactically as sanitizing agent. Said sanitizing agent maybe used before or after surgery, or for example during hemodialysis.Moreover, premature infants and immunocompromised persons, or thosesubjects with need for prosthetic devices may be treated with a modifiedendolysin variant according to the present invention. Said treatment maybe either prophylactically or during acute infection. In the samecontext, nosocomial infections, especially by antibiotic resistantstrains like Pseudomonas aeruginosa (FQRP), Acinetobacter species andEnterobacteriaceae such as E. coli, Salmonella, Shigella, Citrobacter,Edwardsiella, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus,Providencia, Serratia and Yersinia species may be treatedprophylactically or during acute phase with a modified endolysin variantof the present invention. Therefore, a modified endolysin variantaccording to the present invention may be used as a disinfectant also incombination with other ingredients useful in a disinfecting solutionlike detergents, tensids, solvents, antibiotics, lantibiotics, orbacteriocins.

For the use of the modified endolysin variant according to the presentinvention as a disinfectant e.g. in hospital, dental surgery,veterinary, kitchen or bathroom, the modified endolysin variant can beprepared in a composition in form of e.g. a fluid, a powder, a gel, oran ingredient of a wet wipe or a disinfection sheet product. Saidcomposition may additionally comprise suitable carrier, additives,diluting agents and/or excipients for its respective use and form,respectively,—but also agents that support the antimicrobial activitylike EDTA or agents enhance the antimicrobial activity of the fusionproteins. The fusion protein may also be used with common disinfectantagents like, Alcohols, Aldehydes, Oxidizing agents, Phenolics,Quaternary ammonium compounds or UV-light. For disinfecting for examplesurfaces, objects and/or devices the modified endolysin variant can beapplied on said surfaces, objects and/or devices. The application mayoccur for instance by wetting the disinfecting composition with anymeans such as a cloth or rag, by spraying, pouring. The fusion proteinsmay be used in varying concentration depending on the respectiveapplication and the “reaction time” intended to obtain fullantimicrobial activity.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter, however, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description. Itis to be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the invention, as claimed.

The following examples explain the present invention but are notconsidered to be limiting. Unless indicated differently, molecularbiological standard methods were used, as e.g., described by Sambrock etal., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

EXAMPLE 1 Cloning, Expression and Purification of Modified phiKZgp144and ELgpgp188 Endolysin Variants

phiKZgp144 as depicted in SEQ ID NO: 1 and ELgp188 as depicted in SEQ IDNO: 2 are modular endolysins originating from Pseudomonas aeruginosaphages φKZ and EL with an N-terminal peptidoglycan binding andC-terminal catalytic domain (Briers et al., 2007).

For the amplification of the open reading frame (ORF) of phiKZgp144 andELgp188 PCR a standard 5′ primer (for phiKZgp144: 5′ ATGAAAGTATTACGCAAA3′ (SEQ ID NO: 83); for ELgp188 5′ ATGAACTTCCGGACGAAG 3′ (SEQ ID NO:65)) and the standard 3′ primers according to SEQ ID NO: 81 and 82 wereapplied (for phiKZgp144: TTTTCTATGTGCTGCAAC (SEQ ID NO: 81); forELgp188: ATACGAAAT AACGTGACGA (SEQ ID NO: 82)) was used. To extend the5′ end of the open reading frame encoding phiKZgp144 or ELgp188 with agene fragment encoding nine positively charged residues(Lys-Arg-Lys-Lys-Arg-Lys-Lys-Arg-Lys—SEQ ID NO: 11) a tail PCR with anextended 5′ primer (for phiKZgp 144: 5′ ATGGGATCCAAACGCAAGAAACGTAAGAAACGCAAAAAAGTATTACGCAAAG 3′ (SEQ ID NO 79); for ELgp188: 5′ATGGGATCCAAACGCAAGAAACGTAAGAAA CGCAAAAACTTCCGGACGAAG 3′ (SEQ ID NO: 80))and the standard 3′ primers according to SEQ ID NO: 81 and 82 wereapplied. The PCR product was cloned in the pEXP5CT/TOPO® expressionvector (Invitrogen, Carlsbad, Calif., USA) according to the protocol ofthe manufacturer. Arginine triplets were incorporated besides lysinetriplets to avoid tRNA depletion and reduce the risk of frameshifts (theonly two available triplets for lysine are AAA and AAG, leading to longA-stretches). Insertion of additional polycationic cassettes into thedesigned BamHI restriction site lengthens the tail with extra cationicresidues. This insertion creates an arginine and serine triplet at eachjunction site (FIG. 1). Up to four polycationic peptide stretches werefused to both phiKZgp144 and ELgp188, designated (POLY)^(n)-gp144 or(POLY)^(n)-gp188 (n=1,2,3,4), comprising respectively 9, 19, 29 and 39positively charged amino acid residues in the N-terminus. Accordingly,the following constructs were expressed in E. coli BL21 (DE3) pLysScells (exponentially growing cells at 37° C., induction using 1 mM IPTG,expression for 4 h at 37° C.):

Modified endolysin Number of positively variant SEQ ID NO: charged aminoacid residues POLY-gp144 SEQ ID NO: 35 9 (POLY)²-gp144 SEQ ID NO: 36 19(POLY)³-gp144 SEQ ID NO: 37 29 (POLY)⁴-gp144 SEQ ID NO: 38 39 POLY-gp188SEQ ID NO: 39 9 (POLY)²-gp188 SEQ ID NO: 40 19 (POLY)³-gp188 SEQ ID NO:41 29 (POLY)⁴-gp188 SEQ ID NO: 42 39

The modified endolysin variants POLY-gp 144 (SEQ ID NO: 35), (POLY)²-gp144 (SEQ ID NO: 36), POLY-gp188 (SEQ ID NO: 39) and (POLY)²-gp188 (SEQID NO: 40) have been used for further investigations. Said proteins werepurified by Ni²⁺ affinity chromatography using the C-terminal 6× His-tag(Akta Fast Protein Liquid Chromatography using 1 ml His-trap Ni-NTAcolumns). The total yields per liter E. coli expression culture weredetermined by spectrophotometric measurement of the proteinconcentration and the total volume of the purified stock solution. Thepurification of gp188 derivatives was performed under more stringentconditions (65 mM imidazole) compared to gp144 derivatives (50 mMimidazole) to ensure high purity. The total yields per liter E. coliexpression culture are shown in table 1.

TABLE 1 Yields of recombinant purification of endolysin derivatives perliter E. coli expression culture. Endolysin Fusion phiKZgp144 ELgp188POLY   2 mg   48 mg (POLY)² 0.5 mg 0.06 mg

Purified stock solutions were ˜90% pure. Mass spectrometric analysis ofpurified solutions of POLY-derivatives revealed traces of the E. coli50S ribosomal subunit protein L2 and 16S rRNA uridine-516pseudo-uridylate synthase. All phiKZgp144 derivatives showed multimerformation which could be converted to monomers by addition ofβ-mercaptoethanol, indicating that interdisulfide bonds causemultimerization.

EXAMPLE 2 Antibacterial Activity of Modified phiKZgp144 and ELgp188Variants

Exponential (˜10⁶/ml) P. aeruginosa PAO1p cells (Pirnay J P et al.(2003), J Clin Microbiol., 41(3):1192-1202) were 100× diluted (finaldensity was ˜10⁶/ml) and incubated at room temperature with each 10 μgundialyzed protein (unmodified endolysins phiKZgp 144 (SEQ ID NO: 1) andELpg188 (SEQ ID NO: 2) and modified endolysin variants POLY-gp144 (SEQID NO:35), (POLY)²-gp144 (SEQ ID NO: 36), POLY-gp188 (SEQ ID NO: 39) and(POLY)²-gp188 (SEQ ID NO: 40) at a final concentration of 100 μg/ml inbuffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 M imidazole). After 1hour cell suspensions were diluted in PBS buffer (10e-5, 10e-4 and10e-3) and plated (standard LB-medium, incubated overnight at 37° C.).Additionally, a negative control containing cells in PBS buffer wasplated. The residual colonies were counted after an overnightincubation. Based on the counted cell numbers the antibacterial activityas the relative inactivation (%) (=100−(N_(i)/No)*100 with N₀=number ofuntreated cells and N_(i)=number of treated cells) and in logarithmicunits (=log₁₀N₀/N_(i)) was calculated (Table 2). All samples werereplicated in six fold. Averages/standard deviations are represented.Statistical analysis was performed using a student's t-test.

Unmodified endolysins phiKZgp144 and ELgp188 do not reduce cell numberssignificantly compared to the negative control. This observationillustrates the efficacy of the outer membrane as a barrier for theendolysin to degrade the cell wall of the Gram-negative bacteria. Incontrast as shown in Table 2 the incubation with the modified endolysinsPOLY-gp144, (POLY)²-gp144, POLY-gp188 and (POLY)²-gp188 causes asignificant reduction (α=0.05) of the bacterial cell number (99.85±0.09%for POLY-gp144 and 98.0±0.2% for POLY-gp188). An increase of the lengthof the polycationic peptide stretch further tends to strengthen theantibacterial activity, especially in case of phiKZgp144 (a reduction upto 99.98±0.02% or 3.7±0.3 log units is achieved within 1 hour for(POLY)²-gp144). Moreover, the experiments demonstrated that the modifiedendolysins of phiKZgp 144 have a higher antibacterial activity than themodified endolysins of ELgp188.

TABLE 2 Antibacterial effect of endolysins unmodified and modifiedphiKZgp144 and ELgp188 variants. Endolysins Exponentially phiKZgp144ELgp188 growing cells % log % log unmodified  0 ± 15 0.00 ± 0.06  10 ±13 0.05 ± 0.06 endolysin POLY 99.85 ± 0.09 2.9 ± 0.3 98.0 ± 0.2 1.7 ±0.1 (POLY)² 99.98 ± 0.02 3.7 ± 0.3 98.9 ± 0.4 2.0 ± 0.2

Thus, the example demonstrated that the addition of a short peptidestretch of nine cationic residues N-terminally to phiKZgp144 (SEQ IDNO: 1) is already sufficient to kill almost 99.9% of the cells within 1hour. Poly-L-Lysine has intrinsic antibacterial activity as well,although this property is so far only ascribed to polymers of at least20 residues (Vaara and Vaara, 1983a, 1983b). However, the concertedaction of the polycationic peptide stretch and the endolysin kills thecells.

In a further experiment the modified endolysin POLY-gp 144 was dialyzedto 50 mM KH₂P0₄/K₂HP0₄ pH 7 and used instead of undialyzed proteinsolution as described above. Thereby, the inactivation level wasadditionally increased from 2.9±0.3 log units to 3.9±0.2 log units.

EXAMPLE 3 Expression of Modified phiKZgp144 and ELgp188 Variants inPichia pastoris as a Host for Non-Toxic Recombinant Production

The open reading frame encoding POLY-gp144 (SEQ ID NO: 35) was cloned inthe pPICZαA shuttle vector (Invitrogen), which was subsequentlyintegrated in the P. pastoris genome by homologous recombination (asindicated by the manufacturer; P. pastoris X33 cells, Invitrogen). Geneexpression was induced with methanol (1%) in BMMY-medium and thesupernatant was analyzed for the presence of enzymatic activity after 1,3 and 4 days. Therefore, an amount of 30 μl supernatant of the P.pastoris expression culture was added to 270 μl chloroform-permeabilizedP. aeruginosa PAO1p cells (Pirnay JP et al. (2003), J Clin Microbiol.,41(3):1192-1202) after 1, 3 and 4 days (buffer condition: KH₂PO₄/K₂HP0₄I=120 mM pH 6.2). Subsequently, the optical density wasspectrophotometrically recorded (FIG. 2). A drop in optical densityindicates the secretion of a muralytic enzyme by P. pastoris . As anegative control, P. pastoris X33 without expression plasmid wasincluded. Thus, the lysis of the substrate upon addition of thesupernatants sample is a measure for successful recombinant productionand secretion of POLY-gp144 (SEQ ID NO: 35) by P. pastoris . After 1day, a limited enzymatic activity could be detected. The maximumactivity was observed after 3 days since no significant increase ofactivity in the supernatants was observed at the fourth day. No toxiceffect on the cell density of P. pastoris was observed.

During expression by P. pastoris the α-secretion signal of the vectorcauses secretion of the recombinant protein to the surrounding media,which allows a simplify purification since only a limited number ofother proteins is secreted. A BamHI restriction site in the 5′ end ofthe open reading frames enables the addition of more cassettes encodingadditional polycationic peptide stretches.

EXAMPLE 4 Further Modified Endolysin phiKZgp144 Variants with DifferentPolycationic Peptide Stretches

To test and to compare the potential of polycationic peptides variantsof phiKZgp144 and other endolysin encoding genes were synthesised havingdifferent polycationic peptides at the N-terminal end of the protein.Peptide stretch variation concerns length, composition and insertion oflinker sequences. On the one hand further polycationic peptide stretcheshaving N-terminal multiples of the KRK motive were produced. On theother hand polycationic peptide stretches consisting only of arginine(R) or lysine (K) were produced. Moreover, to enhance the translation oflong polycationic peptide stretches, polycationic peptide stretchescomprising a linker sequence were produced.

The different products were cloned in the pET32b expression vector(Novagen, Darmstadt, Germany). pET32b was used to reduce potentialtoxicity of the polycationic peptide against the E. coli host. Avector-encoded fusion protein (thioredoxin) masks the polycationicpeptide and can be eliminated during the purification process.

Accordingly, the following modified endolysin variants were expressed inE. coli BL21 (DE3) cells at 37° C. until an optical density of OD600nm=0.6 was reached. Then protein expression was induced with 1 mM IPTG(final concentration) and expression was preformed for four hours. ThenE. coli cells were harvested by centrifugation for 20 min at 6000 g andcell disruption and protein purification was performed according theS-tag purification kit (Novagen, Darmstadt, Germany):

peptide Modified endolysin stretch's Sequence of the variant lengthpeptide stretch phiKZgp144  0 — (SEQ ID NO: 1) pKKZ144pET32b 10KRKKRKKRKK (SEQ ID NO: 43) (SEQ ID NO: 14) KRK_6_pET32b  6 KRKKRK(SEQ ID NO: 44)   (SEQ ID NO: 10) KRK_12_pET32b 12 KRKKRKKRKKRK(SEQ ID NO: 45) (SEQ ID NO: 15) KRK_14_pET32b 14 KRKKRKKRKKRKKR(SEQ ID NO: 46) (SEQ ID NO: 16) R9_pET32b  9 RRRRRRRRR (SEQ ID NO: 47)(SEQ ID NO: 12) K8_pET32b  8 KKKKKKKK (SEQ ID NO: 48)  (SEQ ID NO: 13)pK2KZ144_pET32b_mod3 38 KRKKRKKRKRGSGSGKRKK (SEQ ID NO: 49)RKKRKGSGSGKRKKRKKRK (SEQ ID NO: 28)

All proteins were purified using the S-Tag™ rEK Purification Kit(Novagen, Darmstadt, Germany). Using the pET32b vector, the expressedproteins were not toxic to the host resulting in high yields of producedprotein. Purified stock solutions showed high purity.

Exponential (˜10⁶/ml) P. aeruginosa PAO1p cells (Burn wound isolate,Queen Astrid Hospital, Brussels; Pirnay J P et al. (2003), J ClinMicrobiol., 41(3):1192-1202) were 100× diluted (final density was˜10⁶/m1) incubated at room temperature with each 10 μg undialyzedprotein as listed above at a final concentration of 100 μg/ml in buffer(20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 M imidazole). After 1 hourcell suspensions were diluted 1:100 and plated on LB. Additionally, anegative control was plated using buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5M NaCl; 0.5 M imidazole). The residual colonies were counted after anovernight incubation at 37° C. Based on the counted cell numbers theantibacterial activity as the relative inactivation (%)(=100−(N_(i)/No)*100 with N₀=number of untreated cells and N_(i)=numberof treated cells) was calculated (Table 3). All samples were replicatedat least in four fold.

TABLE 3 Antibacterial effect of endolysins unmodified andmodified phiKZgp144 and ELgp188 Modified endolysin Sequence of theReduction variant peptide stretch [%] phiKZgp144 0 (SEQ ID NO: 1) pKKZ144pET32b KRKKRKKRKK 99-99.9 (SEQ ID NO: 43) (SEQ ID NO: 14)KRK_6_pET32b KRKKRK 99.9 (SEQ ID NO: 44) (SEQ ID NO: 10) KRK_12_pET32bKRKKRKKRKKRK 99-99.9 (SEQ ID NO: 45) (SEQ ID NO: 15) KRK_14_pET32bKRKKRKKRKKRKKR 99.9 (SEQ ID NO: 46) (SEQ ID NO: 16) R9_pET32b RRRRRRRRR99 (SEQ ID NO: 47) (SEQ ID NO: 12) K8_pET32b KKKKKKKK 99 (SEQ ID NO: 48)(SEQ ID NO: 13) pK2KZ144_pET32b_mod3 KRKKRKKRKRGSGSGKRKK 99.9(SEQ ID NO: 49) RKKRKGSGSGKRKKRKKRK (SEQ ID NO: 28)

Unmodified phiKZgp144 does not reduce cell numbers significantlycompared to the negative control. Beyond that, modified phiKZgp144variants wearing a polycationic peptide of N-terminal multiples of theKRK motive enhance the antimicrobial effect immensely. However, alsovariants having a homomer peptide stretch of lysine or arginine showsignificant reduction of cells compared with unmodified phiKZgp144 asmeasured. Moreover, also the variant having a polycationic peptidestretch of 38 amino acid residues and comprising a linker sequenceenhance the antimicrobial effect immensely.

EXAMPLE 5 Modified Endolysin Variants of Salmonella typhimurium PhagePSP3

PSP3gp10 according to SEQ ID NO: 8 is a globular endolysin with 165amino acid residues originating from Salmonella typhimurium phage PSP3with a catalytic lambda-like muramidase domain. As predicted by BLASTpand Pfam analysis the PSP3gp10 endolysin comprises its catalytic domainin the range of about amino acid residue 34 to about amino acid residue152.

Purified genomic DNA of phage PSP3 was used as a template for theamplification of the open reading frame (ORF) of PSP3gp10 in a Hot StartTaq polymerase PCR reaction (Qiagen, Germany) using the following PCRparameters:

For said PCR a standard 5′ primer (5′ ATGGGATCCCCGGTCATTAATACTCACCAG 3′(SEQ ID NO: 50)) and a standard 3′ primer (5′ TGCCATCACCCCGCCAGCCGTG 3′(SEQ ID NO: 51)) was used. To extend the 5′ end of the ORF which encodesPSP3gp10 with a gene fragment encoding the polycationic 9-mer peptideLys-Arg-Lys-Lys-Arg-Lys-Lys-Arg-Lys (SEQ ID NO: 11) a tail PCR (HotStart Taq polymerase PCR with same parameters) with an extended 5′primer (5′ ATGGGATCCAAACGCAAGAAACGTAA GAAACGCAAACCGGTCATTAATACTCACCAG 3′(SEQ ID NO: 52)) and the standard 3′ primer according to SEQ ID NO: 51was applied. Both the original unmodified PSP3gp10 PCR fragment and thePK-extended fragment were ligated in the pEXP5CT/TOPO® expression vector(Invitrogen, Carlsbad, Calif., USA) by following the TA-cloning protocolof the manufacturer.

Recombinant expression of PSP3gp10 according to SEQ ID NO: 8 andPKPSP3gp10 according to SEQ ID NO: 53 is performed in exponentiallygrowing E. coli BL21 (λDE3) pLysS cells (Invitrogen) after inductionwith 1 mM IPTG (isopropylthiogalactoside) at 37° C. for a period of 4hours. Both proteins were purified by Ni²⁺ affinity chromatography (AktaFPLC, GE Healthcare) using the C-terminal 6× His-tag, encoded by thepEXP5CT/TOPO® expression vector. The Ni²⁺ affinity chromatography isperformed in 4 subsequent steps, all on room temperature:

-   -   1 . Equilibration of the Histrap HP 1 ml column (GE Healthcare)        with 10 column volumes of Washing Buffer (60 mM imidazole, 0.5        mM NaCl and 20 mM NaH₂P0₄-NaOH on pH 7.4) at a flow rate of 0.5        ml/min.    -   2 . Loading of the total lysate (with wanted endolysin) on the        Histrap HP 1 ml column at a flow rate of 0.5 ml/min.    -   3 . Washing of the column with 15 column volumes of Washing        Buffer at a flow rate of 1 ml/min.    -   4 . Elution of bounded endolysin from the column with 10 column        volumes of Elution Buffer (500 mM imidazole, 5 mM NaCl and 20 mM        NaH₂PO₄-NaOH on pH 7.4) at a flow rate of 0.5 ml/min

The total yields of both purified recombinant proteins per liter E. coliexpression culture shown in Table 4 . The values were determined byspectrophotometric measurement of the protein concentration and thetotal volume of the purified stock solution at a wavelength of 280 nm.Purified stock solutions consisting of PSP3gp10 and PKPSP3gp10,respectively, in Elution Buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl;500 mM imidazole) were at least 90% pure as determined visually onSDS-PAGE gels.

TABLE 4 Yields of purified recombinant PSP3gp10 endolysin and itsmodified variant PKPSP3gp10 per liter E. coli expression culture.Endolysins Expression yield PSP3gp10 (SEQ ID NO: 8) 2.15 mg PKPSP3gp10(SEQ ID NO: 53) 5.56 mg

To determine the anti-Gram-negative spectrum of the PKPSP3gp10 endolysinaccording to SEQ ID NO: 53, a combination of 1.315 μM PKPSP3gp10endolysin and 0.5 mM EDTA was tested on the clinical P. aeruginosastrains PAO1p and Br667, Escherichia coli WK6, and Salmonellatyphimurium (see Table 5). Exponential growing bacterial cells(OD_(600 nm) of 0.6) were 100-fold diluted to a final density of about10⁶/ml of each strain were incubated for 30 minutes at room temperaturewithout shaking with unmodified endolysin PSP2gp10 (SEQ ID NO: 8) andmodified endolysin PKPSP3gp10 (SEQ ID NO: 53) each in combinationwithout and with 0.5 mM EDTA. For incubation, the endolysins were usedeach in buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 M imidazole)and the incubation took place at a final concentration of endolysin of1.315 μM. As a control each strain was also incubated for 30 minuteswith 0.5 mM EDTA (in same buffer as outlined above) but no endolysin.

TABLE 5 List of used Gram-negative strains Gram-negative strain SourceReference Pseudomonas aeruginosa Burn wound isolate, Queen Pirnay etal., PAO1p Astrid Hospital, Brussels 2003* Pseudomonas aeruginosa Burnwound isolate, Queen Pirnay et al., Br667 Astrid Hospital, Brussels2003* Escherichia coli WK6 Standard laboratory Prof. C. Michielsexpression strain Salmonella typhimurium SGSC N° 2317 Prof. C. MichielsLT2 *Pirnay J P et al. (2003). Molecular epidemiology of Pseudomonasaeruginosa colonization in a burn unit: persistence of amultidrug-resistant clone and a silver sulfadiazine-resistant clone. JClin Microbiol., 41(3): 1192-1202.

After incubation cell suspensions were diluted three times (respectively10⁵-10⁴-10³ cells/ml) and 100 μl of each dilution was plated out onLB-medium. The residual colonies were counted after an overnightincubation on 37° C. Based on the counted cell numbers the antibacterialactivity as the relative inactivation in logarithmic units(=log₁₀N₀/N_(i) with N₀=number of untreated cells and N_(i)=number oftreated cells) was calculated (Table 6).

TABLE 6 Antibacterial activity of unmodified endolysin (PSP3gp10) andits modified endolysin variant (PKPSP3gp10) with and without EDTA-Na₂ ondifferent exponential growing Gram-negative species. 1.315 μM 1.315 μM1.315 μM 1.315 μM PSP3gp10 + PKPSP3gp10 + 0.5 mM EDTA PSP3gp10PKPSP3gp10 0.5 mM EDTA 0.5 mM EDTA P. aeruginosa 0.146 +/− 0.002 0.383+/− 0.015 0.344 +/− 0.163 3.552 +/− 0.536 >4.146 PAO1p P. aeruginosa0.223 +/− 0.038 0.375 +/− 0.056 0.353 +/− 0.086 0.571 +/− 0.035 0.891+/− 0.118 Br667 Salmonella 0.104 +/− 0.049 0.283 +/− 0.038 0.327 +/−0.057 0.690 +/− 0.036 0.850 +/− 0.032 typhimurium Escherichia coli 0.393+/− 0.035 0.190 +/− 0.029 0.205 +/− 0.088 0.387 +/− 0.014 0.584 +/−0.024 WK6

All samples were replicated in threefold. Averages+/−standard deviationsare represented. The maximal reduction observed is dependent on thedetection level of 10 cells/ml and the initial cell density. For PAO1p,EDTA works synergistically with both the unmodified PSP3gp10 endolysinand its modified variant PKPSP3gp10.

EXAMPLE 6 Modified Endolysin Variants of Escherichia coli Phage P2

P2gp09 according to SEQ ID NO: 9 is a globular endolysin of 165 aminoacid residues originating from Escherichia coli phage P2 with acatalytic lambda-like muramidase domain. As predicted by BLASTp and Pfamanalysis the P2gp09 endolysin comprises its catalytic domain in therange of about amino acid residue 34 to about amino acid residue 152.

Purified genomic DNA of phage P2 was used as a template for theamplification of the open reading frame (ORF) of P2gp09 in standard PCRreaction with Pfu polymerase (Fermentas) using the following PCRparameters:

For said PCR a standard 5′ primer (5′ ATGGGATCCCCGGTAATTAACACGCATC 3′(SEQ ID NO: 54)) and a standard 3′ primer (5′ AGCCGGTACGCCGCCAGCGGTACGC3′ (SEQ ID NO: 55)) was used. To extend the 5′ end of the ORF whichencodes P2gp09 with a gene fragment encoding the polycationic 9-merpeptide Lys-Arg-Lys-Lys-Arg-Lys-Lys-Arg-Lys (SEQ ID NO: 11) a tail PCR(with same parameters as standard PCR above) with an extended 5′ primer(5′ ATGGGATCCAAACGCAAGAAACGTAAGAAACGC AAACCGGTAATTAACACGCATC 3′ (SEQ IDNO: 56) and the standard 3′ primer according to SEQ ID NO 55 wasapplied. Both the original unmodified P2gp09 PCR fragment and theextended fragment were ligated in the pEXP5CT/TOPO® expression vector(Invitrogen, Carlsbad, Calif., USA) by following the TA-cloning protocolof the manufacturer.

Recombinant expression of P2gp09 according to SEQ ID NO: 9 and PKP2gp09according to SEQ ID NO: 57 is performed in exponentially growing E. coliBL21 (λDE3) pLysS cells (Invitrogen) after induction with 1 mM IPTG(isopropylthiogalactoside) at 37° C. for a period of 4 hours. Bothproteins were purified by Ni²⁺ affinity chromatography (Akta FPLC, GEHealthcare) using the C-terminal 6× His-tag, encoded by thepEXP5CT/TOPO® expression vector. The Ni²⁺ affinity chromatography isperformed in 4 subsequent steps, all on room temperature:

-   -   1 . Equilibration of the Histrap HP 1 ml column (GE Healthcare)        with 10 column volumes of Washing Buffer (60 mM imidazole, 0.5        mM NaCl and 20 mM NaH₂P0₄-NaOH on pH 7.4) at a flow rate of 0.5        ml/min.    -   2 . Loading of the total lysate (with wanted endolysin) on the        Histrap HP 1 ml column at a flow rate of 0.5 ml/min.    -   3 . Washing of the column with 15 column volumes of Washing        Buffer at a flow rate of 1 ml/min.

4 . Elution of bounded endolysin from the column with 10 column volumesof Elution Buffer (500 mM imidazole, 5 mM NaCl and 20 mM NaH₂P0₄-NaOH onpH 7.4) at a flow rate of 0.5 ml/min

The total yields of both purified recombinant proteins per liter E. coliexpression culture shown in Table 7 . The values were determined byspectrophotometric measurement of the protein concentration and thetotal volume of the purified stock solution at a wavelength of 280 nm.Purified stock solutions consisting of P2gp09 and PKP2gp09,respectively, in Elution Buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl;500 mM imidazole) were at least 95% pure as determined visually onSDS-PAGE gels.

TABLE 7 Yields of purified recombinant P2gp09 endolysin and itsPK-modified derivative PKP2gp09 per liter E. coli expression culture.Endolysins Expression yield P2gp09 (SEQ ID NO: 9) 5.52 mg PKP2gp09 (SEQID NO: 57) 3.40 mg

To determine the anti-Gram-negative spectrum of the PK2gp09 endolysinaccording to SEQ ID NO: 57, a combination of 1.315 μM PK2gp09 endolysinand 0,5 mM EDTA was tested on the clinical P. aeruginosa strains PAO1pand Br667 and on Escherichia coli WK6 (see Table 9). Exponential growingbacterial cells (OD_(600 nm) of 0.6) were 100-fold diluted to a finaldensity of about 10⁶/ml of each strain was incubated for 30 minutes atroom temperature without shaking with unmodified endolysin P2gp09 (SEQID NO: 9) and modified endolysin PKP2gp09 (SEQ ID NO: 57) each incombination without and with 0.5 mM EDTA. For incubation, the endolysinswere used each in buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 Mimidazole) and the incubation took place at a final concentration ofendolysin of 1.315 μM. As a control each strain was also incubated for30 minutes with 0.5 mM EDTA (in same buffer as outlined above) but noendolysin. After incubation cell suspensions were diluted three times(respectively 10⁵-10⁴-10³ cells/ml) and 100 μl of each dilution wasplated out on LB-medium. The residual colonies were counted after anovernight incubation on 37° C. Based on the counted cell numbers theantibacterial activity as the relative inactivation in logarithmic units(=log₁₀N₀/N_(i) with N₀=number of untreated cells and N_(i)=number oftreated cells, both counted after incubation) was calculated (Table 8).

TABLE 8 Antibacterial activity of unmodified endolysin (P2gp09) and itsmodified endolysin variant (P2gp09) with and without EDTA-Na₂ ondifferent exponential growing Gram- negative species. 1.315 μM P2gp09 +1.315 μM 0.5 mM 1.315 μM 1.315 μM 0.5 mM PKP2gp09 + EDTA P2gp09 PKP2gp09Δ EDTA 0.5 mM EDTA Δ P. aeruginosa 0.330 +/− 0.374 +/− 0.326 +/− 0.069−0.038 2.840 +/− 3.172 +/− 0.056 0.332 PAO1p 0.146 0.084 0.079 P.aeruginosa 0.003 +/− 0.246 +/− 0.300 +/− 0.062 0.054 0.582 +/− 0.952 +/−0.213 0.370 Br667 0.051 0.042 0.074 P. putida G1 0.072 +/− 0.419 +/−1.014 +/− 0.139 0.595 3.919 +/− >4,386 >0.467 0.084 0.024 0.118Burkholderia 0.206 +/− 0.769 +/− 1.163 +/− 0.073 0.394 3.8909 +/− 4.255+/− 0.001 0.365 pseudomallei 0.151 0.110 0.056 Escherichia coli 0.153+/− 0.751 +/− 1.104 +/− 0.039 0.353 0.784 +/− 1.545 +/− 0.102 0.749 WK60.046 0.053 0.071

All samples were replicated in threefold. Averages+/−standard deviationsare represented. The maximal reduction observed is dependent on thedetection level of 10 cells/ml and the initial cell density.

TABLE 9 List of used Gram-negative strains Gram-negative strain SourceReference Pseudomonas aeruginosa Burn wound isolate, Queen Pirnay etal., PAO1p Astrid Hospital, Brussels 2003* Pseudomonas aeruginosa Burnwound isolate, Queen Pirnay et al., Br667 Astrid Hospital, Brussels2003* Burkholderia Clinical isolate, UZ Prof J. Verhaegen pseudomalleiGasthuisberg, Leuven Escherichia coli WK6 Standard laboratory Prof C.Michiels expression strain Pseudomonas putida G1 Soil isolate, MoskowProf V. Krylov *Pirnay J P et al., (2003). Molecular epidemiology ofPseudomonas aeruginosa colonization in a burn unit: persistence of amultidrug-resistant clone and a silver sulfadiazine-resistant clone. JClin Microbiol., 41(3): 1192-1202.

EXAMPLE 7 Modified Endolysin Variants of Pseudomonas putida Phage OBP

OBPgpLYS according to SEQ ID NO: 7 is a modular endolysin of 328 aminoacid residues originating from Pseudomonas putida phage OBP with aputative N-terminal peptidoglycan binding domains and a C-terminalcatalytic chitinase domain. As predicted by BLASTp and Pfam analysis theOBPgpLYS endolysin comprises its catalytic domain in the range of aboutamino acid residue 126 to about amino acid residue 292 and theN-terminal peptidoglycan binding domain in the range of about amino acidresidues 7 to 96.

Purified genomic DNA of phage OBP was used as a template for theamplification of the open reading frame (ORF) of OBPgpLYS in standardPCR reaction with Pfu polymerase (Fermentas, Ontario, Canada) using thefollowing PCR parameters:

Therefore a standard 5′ primer (5′ ATGAAAAATAGCGAGAAGAAT 3′ (SEQ ID NO:58)) and a standard 3′ primer (5′ AACTATTCCGAGTGCTTTCTTTGT 3′ (SEQ IDNO: 59)) was used. To extend the 5′ end of the ORF which encodesOBPgpLYS with a gene fragment encoding the polycationic 9-mer peptideLys-Arg-Lys-Lys-Arg-Lys-Lys-Arg-Lys- (SEQ ID NO: 11) a tail PCR (withsame parameters as standard PCR above) with an extended 5′ primer (5′ATGGGATCCAAACGCAAGAAACGTAAGAAACGCAAAAAAAATAGCGAG AAGAAT 3′ (SEQ ID NO:60)) and the standard 3′ primer according to SEQ ID NO 59 was applied.Both the original unmodified OBPgpLYS PCR fragment and the extendedfragment were ligated in the pEXP5CT/TOPO® expression vector(Invitrogen, Carlsbad, Calif., USA) by following the TA-cloning protocolof the manufacturer.

Recombinant expression of OBPgpLYS according to SEQ ID NO: 7 andPKOBPgpLYS according to SEQ ID NO: 61 is performed in exponentiallygrowing E. coli BL21 (λDE3) pLysS cells (Invitrogen) after inductionwith 1 mM IPTG (isopropylthiogalactoside) at 37° C. for a period of 4hours. Both proteins were purified by Ni²⁺ affinity chromatography (AktaFPLC, GE Healthcare) using the C-terminal 6× His-tag, encoded by thepEXP5CT/TOPO® expression vector. The Ni²⁺ affinity chromatography isperformed in 4 subsequent steps, all on room temperature:

-   -   1 . Equilibration of the Histrap HP 1 ml column (GE Healthcare)        with 10 column volumes of Washing Buffer (60 mM imidazole, 0.5        mM NaCl and 20 mM NaH₂P0₄-NaOH on pH 7.4) at a flow rate of 0.5        ml/min.    -   2 . Loading of the total lysate (with wanted endolysin) on the        Histrap HP 1 ml column at a flow rate of 0.5 ml/min.    -   3 . Washing of the column with 15 column volumes of Washing        Buffer at a flow rate of 1 ml/min.    -   4 . Elution of bounded endolysin from the column with 10 column        volumes of Elution Buffer (500 mM imidazole, 5 mM NaCl and 20 mM        NaH₂P0₄-NaOH on pH 7.4) at a flow rate of 0.5 ml/min

The total yields of both purified recombinant proteins per liter E. coliexpression culture shown in Table 10 . The values were determined byspectrophotometric measurement of the protein concentration and thetotal volume of the purified stock solution at a wavelength of 280 nm.Purified stock solutions consisting of OBPgpLYS and PKOBPgpLYS,respectively, in Elution Buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl;500 mM imidazole) were at least 90% pure as determined visually onSDS-PAGE gels.

TABLE 10 Yields of purified recombinant OBPgpLYS endolysin and itsPK-modified derivative PKOBPgpLYS per liter E. coli expression culture.Endolysins Expression yield OBPgpLYS (SEQ ID NO: 7) 3.3 mg PKOBPgpLYS(SEQ ID NO: 61) 4.7 mg

To determine the anti-Gram-negative spectrum of the PKOBPgpLYS endolysinaccording to SEQ ID NO: 61, a combination of 1.313 μM PK OBPgpLYSendolysin and 0.5 mM EDTA was tested on the clinical multiresistant P.aeruginosa strain Br667, Pseudomonas putida G1 (host of phage OBP) and arange of other Gram-negative pathogens (Escherichia coli WK6, Salmonellatyphimurium LT2 and Burkholderia pseudomallei) (see Table 12).Exponential growing bacterial cells (OD_(600 nm) of 0.6) were 100-folddiluted to a final density of about 10⁶/ml of each strain was incubatedfor 30 minutes at room temperature without shaking with unmodifiedendolysin OBPgpLYS (SEQ ID NO: 7) and modified endolysin PKOBPgpLYS (SEQID NO: 61) each in combination without and with 0.5 mM EDTA. Forincubation, the endolysins were used each in buffer (20 mM NaH₂P0₄-NaOHpH7.4; 0.5 M NaCl; 0.5 M imidazole) and the incubation took place at afinal concentration of endolysin of 1.313 μM. As a control each strainwas also incubated for 30 minutes with 0.5 mM EDTA (in same buffer asoutlined above) but no endolysin. After incubation cell suspensions werediluted three times (respectively 10⁵-10⁴-10³ cells/ml) and 100 μl ofeach dilution was plated out on LB-medium. The residual colonies werecounted after an overnight incubation on 37° C. Based on the countedcell numbers the antibacterial activity as the relative inactivation inlogarithmic units (=log₁₀N₀/N_(i) with N₀=number of untreated cells andN_(i)=number of treated cells, both counted after incubation) wascalculated (Table 11). All samples were replicated in threefold.Averages+/−standard deviations are represented. The maximal reductionobserved is dependent on the detection level of 10 cells/ml and theinitial cell density.

TABLE 11 Antibacterial activity of unmodified endolysin (OBPgpLYS) andits modified endolysin variant (PKOBPgpLYS) with and without EDTA-Na₂ ondifferent exponential growing Gram-negative species. 1.313 μM 1.313 μM1.313 μM 1.313 μM OBPgpLYS + PKOBPgpLYS + 0.5 mM EDTA OBPgpLYSPKOBPgpLYS 0.5 mM EDTA 0.5 mM EDTA P. aeruginosa 0.130 +/− 0.023 2.531+/− 0.173 3.079 +/− 0.015 4.357 +/− 1.857 >5.687 PAO1p P. aeruginosa0.031 +/− 0.023 1.082 +/− 0.083 1.163 +/− 0.063 3.144 +/− 0.223 5.272+/− 0.573 Br667 P. putida G1 0.412 +/− 0.055 0.141 +/− 0.027 0.904 +/−0.079 4.891 +/− 0.000 >4.891 Burkholderia 0.220 +/− 0.081 0.997 +/−0.131 1.806 +/− 0.287  4.08 +/− 0.301 >4.861 pseudomallei Escherichiacoli 0.592 +/− 0.113 0.681 +/− 0.032 1.434 +/− 0.018 1.179 +/− 0.2001.695 +/− 0.147 WK6 Salmonella 0.054 +/− 0.048 0.076 +/− 0.011 0.127 +/−0.013 0.774 +/− 0.052 0.908 +/− 0.037 typhimurium

TABLE 12 List of used Gram-negative strains Gram-negative strain SourceReference Pseudomonas aeruginosa Burn wound isolate, Queen Pirnay etal., PAO1p Astrid Hospital, Brussels 2003* Pseudomonas aeruginosa Burnwound isolate, Queen Pirnay et al., Br667 Astrid Hospital, Brussels2003* Pseudomonas putida G1 Soil isolate, Moskow Prof V. KrylovBurkholderia Clinical isolate, UZ Prof J. Verhaegen pseudomalleiGasthuisberg, Leuven Escherichia coli WK6 Standard laboratory Stratageneexpression strain Salmonella typhimurium SGSC N° 2317 Prof C. MichielsLT2 *Pirnay J P, De Vos D, Cochez C, Bilocq F, Pirson J, Struelens M,Duinslaeger L, Cornelis P, Zizi M, Vanderkelen A. (2003). Molecularepidemiology of Pseudomonas aeruginosa colonization in a burn unit:persistence of a multidrug-resistant clone and a silversulfadiazine-resistant clone. J Clin Microbiol., 41(3): 1192-1202.

While the global efficacy of the OBPgpLYS treatment is speciesdependent, the results in table 11 show an added effect of thePKOBPgpLYS compared to unmodified OBPgpLYS for all bacterial speciestested, both in the absence as the presence of 0.5 mM EDTA. ForPseudomonas and Burkholderia species, a clear synergistic effect withEDTA is observed for the PKOBPgpLYS activity.

EXAMPLE 8 Effect of Different EDTA Concentration on the AntibacterialActivity of OBPgpLYS and PKOBPgpLYS

To determine the influence of EDTA on the antibacterial activity ofunmodified and modified endolysins the antibacterial activity of theunmodified OBPgpLYS endolysin (SEQ ID NO: 7) and the PKOBPgpLYSendolysin (SEQ ID NO: 61) was tested on Pseudomonas aeruginosa PAO1pcells (Pirnay J P et al. J Clin Microbiol., 41(3):1192-1202 (2003))using different concentrations of EDTA and endolysins. Exponentialgrowing bacterial cells (OD_(600 nm) of 0.6) were 100-fold diluted to afinal density of about 10⁶/ml and incubated for 30 minutes at roomtemperature without shaking with unmodified endolysin OBPgpLYS (SEQ IDNO: 7) and modified endolysin PKOBPgpLYS (SEQ ID NO: 61). Forincubation, the endolysins were used each in buffer (20 mM NaH₂P0₄-NaOHpH7.4; 0.5 M NaCl; 0.5 M imidazole) at final concentrations of endolysinof 0.013 μM, 0.131 μM and 1.315 μM. Thereby, the following differentEDTA concentrations were used: 0 mM, 0.05 mM, 0.5 mM and 10 mM. As acontrol one sample was also incubated for 30 minutes with no endolysin,instead of there was buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 Mimidazole) added. After incubation cell suspensions were diluted threetimes (respectively 10⁵-10⁴-10³ cells/ml) and 100 μl of each dilutionwas plated out on LB-medium. The residual colonies were counted after anovernight incubation on 37° C. Based on the counted cell numbers theantibacterial activity as the relative inactivation in logarithmic units(=log₁₀N₀/N_(i) with N₀=number of untreated cells and N_(i)=number oftreated cells, both counted after incubation) was calculated (Table 13).All samples were replicated in threefold. Averages+/−standard deviationsare represented. The maximal reduction observed (5.69 log units) isdependent on the detection level of 10 cells/ml and the initial celldensity. “Δ” gives the difference of activity between the respectiveOBPgpLYS and PKOBPgpLYS samples.

TABLE 13 Antibacterial activity of unmodified endolysin (OBPgpLYS) andits modified endolysm variant (PKOBPgpLYS) in combination with differentEDTA concentrations on exponential growing Pseudomonas aeruginosa PAO1pcells Concentration of EDTA-Na₂ (in mM) 0 0.05 0.5 10 No endolysin /0.028 +/− 0.008 0.130 +/− 0.023 1.827 +/− 0.052 0.013 μM OBPgpLYS 0.956+/− 0.110 / 4.626 +/− 0.287 / 0.013 μM PKOBPgpLYS 0.992 +/− 0.181 /5.204 +/− 0.000 / Δ 0.036   0.578 0.131 μM OBPgpLYS 2.158 +/− 0.027 /4.599 +/− 0.275 / 0.131 μM PKOBPgpLYS 2.529 +/− 0.184 / 5.671 +/− 0.000/ Δ 0.371   1.072 1.315 μM OBPgpLYS 2.531 +/− 0.173 2.762 +/− 0.0914.357 +/− 1.857 4.888 +/− 0.275 1.315 μM PKOBPgpLYS 3.079 +/− 0.0154.145 +/− 0.015 >5.687 >5.687 Δ 0.548 1.383 >1.330 >0.799

As shown in Table 13 unmodified endolysin OBPgpLYS reduces cell numberssignificantly with more than 2.5 log units for 1.315 μM and with +/− 1log unit for 0.013 μM, compared to the negative control. Modifiedendolysin PKOBPgpLYS results in an added 0.5 log units reduction forexponentially growing PAO1p cells. The observed antibacterial effect canbe increased to more as 5.69 log units reduction (beneath the detectionlevel) by combining PKOBPgpLYS with the outer membrane permeabilizerEDTA-Na₂ at a concentration of 0.5 and 10 mM EDTA. The difference inactivity between the unmodified OBPgpLYS and the PK-modified OBPgpLYSincreases by raising the amount of added endolysin (from 0.013-1.315 μMendolysin).

EXAMPLE 9 Antibacterial Activity of Modified phiKZgp144 Variants onDifferent Gram-Negative Bacteria

To test and to compare the potential of polycationic peptides variantsof phiKZgp144 and other endolysins, encoding genes were synthesisedhaving polycationic peptides at the N-terminal end of the protein.

The different products were cloned in the pET32b expression vector(Novagen, Darmstadt, Germany). pET32b was used to reduce potentialtoxicity of the polycationic peptide against the E. coli host. Avector-encoded fusion protein (thioredoxin) masks the polycationicpeptide and can be eliminated during the purification process.

The genes encoding smi01 (YP_001712536) and KRK9_smi01 (SEQ ID NO: 75)were fully synthesised (Entelechon, Regensburg, Germany) and cloned intopET32b.

Accordingly, the following modified endolysin variants were expressed inE. coli BL21 (DE3) cells at 37° C. until an optical density of OD600nm=0.6 was reached: smi01 (YP_001712536), KRK9_smi01 (SEQ ID NO: 75),phiKZgp144 (SEQ ID NO: 1), pKKZ144pET32b (SEQ ID NO: 43) and POLYKZ144(SEQ ID NO: 35). Protein expression was induced with 1mM IPTG (finalconcentration) and expression was preformed for four hours. Then E. colicells were harvested by centrifugation for 20 min at 6000 g and celldisruption and protein purification was performed using the S-Tag™ rEKPurification Kit (Novagen, Darmstadt, Germany). Using the pET32b vector,the expressed proteins were not toxic to the host resulting in highyields of produced protein. Purified stock solutions showed high purity.

For testing and as reference for comparison phiKZgp144 and POLYgp144were synthesized and purified as described in EXAMPLE 1.

Exponential (˜10⁶/ml) growing cells of P. aeruginosa PAO1p (Burn woundisolate, Queen Astrid Hospital, Brussels; Pirnay J P et al. (2003), JClin Microbiol., 41(3):1192-1202), Acinetobacter baumanii (DSMZ 30007)or Burkholderia solanaceum (Isolate provided by Prof. C. Michiels) were100× diluted (final density was ˜10⁶/ml) incubated at room temperaturewith each 10 μg undialyzed protein as listed above at a finalconcentration of 100 μg/ml in buffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 MNaCl; 0.5 M imidazole). After 1 hour cell suspensions were diluted 1:100and plated on LB. Additionally, a negative control was plated usingbuffer (20 mM NaH₂P0₄-NaOH pH7.4; 0.5 M NaCl; 0.5 M imidazole). Theresidual colonies were counted after an overnight incubation at 37° C.Based on the counted cell numbers the antibacterial activity as therelative inactivation (%) (=100−(N_(i)/No)*100 with N₀=number ofuntreated cells and N_(i)=number of treated cells) was calculated (Table3). All samples were replicated at least in four fold.

TABLE 14 Antibacterial effect of different modified endolysin variants(NCBI numbers in brackets) on different bacterial species ReductionProtein Bacterial species [%] smi01 Acinetobacter baumannii DSMZ 30007 0(YP_001712536) KRK9_smi01 Acinetobacter baumannii DSMZ 30007 50phiKZgp144 Pseudomonas aeruginosa 0 pKKZ144pET32b Pseudomonas aeruginosa99-99.9 phiKZgp144 Acinetobacter baumannii DSMZ 30007 0 pKKZ144pET32bAcinetobacter baumannii DSMZ 30007 99.9 phiKZgp144 Burkholderiasolanacearum 0 POLYKZ144 Burkholderia solanacearum 99-99.9

Unmodified endolysins phiKZgp144 and smi01 (YP_001712536) do not reducecell numbers significantly compared to the negative control. Thisobservation again illustrates the efficacy of the outer membrane as abarrier for the endolysin to degrade the cell wall of the Gram-negativebacteria. In contrast as shown in Table 14 the incubation with themodified endolysins KRK9_smi01, pKKZ144pET32b and POLY-gp144 causes asignificant reduction of the bacterial cell number on Acinetobacterbaumanii (50% for KRK_smi01; 99.9% for pKKZ144pET32b), Pseudomonasaeruginosa (90-99.9% for pKKZ144pET32b) and Burkholderia solanaceum(90-99.9% for POLYKZ144).

These experiments demonstrate the applicability of thecationic/polycationic fusion approach for other endolysins. Moreover,the experiments demonstrated that the modified endolysins are active ona variety of bacteria.

The invention claimed is:
 1. An endolysin variant comprising anbacteriophage peptidoglycan hydrolase to which a cationic peptidestretch with LPS disrupting activity is fused, wherein said cationicpeptide stretch consists of 5 to 50 amino acid residues and wherein atleast 70% of the amino acid residues comprised in said peptide stretchare arginine and/or lysine and 0% to 30% are serine and/or glycine, andwherein said endolysin variant exhibits (i) the activity of degrading acell wall of Gram-negative bacteria and (ii) LPS disrupting activity. 2.The endolysin variant according to claim 1, wherein said cationicpeptide stretch consists of 5 to 30 amino acid residues.
 3. Theendolysin variant according to claim 1, wherein said cationic peptidestretch is fused to the N- and/or the C-terminus of the endolysin, inparticular to the N-terminus of the endolysin.
 4. The endolysin variantaccording to claim 1, wherein said endolysin is selected from the groupconsisting of phiKZgp144 according to SEQ ID NO:1, ELgp188 according toSEQ ID NO:2, Salmonella endolysin according to SEQ ID NO:3,Enterobacteria phage T4 endolysin according to SEQ ID NO:4,Acinetobacter baumanii endolysin according to SEQ ID NO:5, E.coli PhageK1F endolysin according to SEQ ID NO:6, PSP3 salmonella endolysinaccording to SEQ ID NO: 8 and E. coli Phage P2 endolysin according toSEQ ID NO:
 9. 5. The endolysin variant according to claim 1, whereinsaid cationic peptide stretch comprises at least one KRK motif, inparticular wherein said peptide stretch consists of a sequence selectedfrom the group consisting of SEQ ID: 10 to
 30. 6. The endolysin variantaccording to claim 1, wherein said endolysin variant comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 35 to 49,53, 57, 62 to 64 and 66 to
 78. 7. An isolated nucleic acid moleculecomprising a nucleotide sequence coding an endolysin variant accordingto claim
 1. 8. A vector comprising the nucleic acid molecule accordingto claim
 7. 9. A host cell comprising a vector according to claim 8,wherein said host cell is in particular a bacterial cell or a yeastcell.
 10. A method for the production of an endolysin variant comprisingculturing a host cell according to claim 9 under conditions supportingthe expression of said endolysin variant.
 11. A method of treating aGram-negative bacterial infection in a subject comprising administeringto said subject an endolysin variant according to claim
 1. 12. A methodfor the removal, reduction and/or prevention of Gram-negative bacterialcontamination of foodstuff, of food processing equipment, of foodprocessing plants, of surfaces coming into contact with foodstuff, ofmedical devices, of surfaces in hospitals and surgeries comprisingcontacting said foodstuff, food processing equipment, food processingplants, surfaces coming into contact with foodstuff, medical devices, orsurfaces in hospitals with an endolysis variant according to claim 1.13. A method for detecting bacterium in a sample from a subject or in afood or feed, or environmental sample comprising contacting said asample with an endolysin variant according to claim
 1. 14. Apharmaceutical composition comprising an endolysin variant according toclaim
 1. 15. The endolysin variant according to claim 1, wherein thecell wall of the Gram-negative bacteria selected from the groupconsisting of Enterobacteriaceae, in particular Escherichia, Salmonella,Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella,Morganella, Proteus, Providencia, Serratia,and Yersinia,Pseudomonadaceae, in particular Pseudomonas, Burkholderia,Stenotrophomonas, Shewanella, Sphingomonas and Comamonas, Neisseria,Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella,Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia,Actinobacillus, Gardnerella, Spirochaetaceae, in particular Treponemaand Borrelia, Leptospiraceae, Campylobacter, Helicobacter, Spirillum,Streptobacillus, Bacteroidaceae, in particular Bacteroides,Fusobacterium, Prevotella and Porphyromonas, and Acinetobacter, inparticular A. baumanii.